Adjuvant-type immunogenic compositions against group B Neisseria meningitidis

Abstract

The present disclosure relates to 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, and an aluminum hydroxyphosphate (AlPO4) adjuvant selected as having a point of zero charge (PZC) of less than 5.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 370,333, filed August 3, 2022, which is incorporated by reference herein in its entirety for all purposes.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, which is 14,094 bytes in size and is provided as a file entitled "PR95421_WO_S17_SANOFI_PASTEUR" created on July 21, 2023. The information in this electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to an AIPO4-adjuvanted immunogenic composition against Neisseria meningitidis group B and methods and uses for stabilizing antigens with adjuvants and enhancing immunogenic responses to autoantigens and heterologous antigens. [Background technology]

[0004] Neisseria meningitidis is a Gram-negative diplococcus whose only known natural host is humans. N. meningitidis is a frequent colonizer of the human nasopharynx and oropharynx, but can also be found in other body regions, 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] Based on the immunochemistry of the capsular polysaccharide (PS), meningococci have been classified into at least 12 different serogroups. 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 has been implicated in local epidemics and some outbreaks (Harrison et al., [ed.] Orenstein WA, Offit PA, Edwards KM, Plotkin SA. Vaccines. 7. Philadelphia (PA): Elsevier; 2018. p. 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 (N. meningitidis) serogroup B is a respiratory bacterium (droplet-transmitted) that cannot survive in the environment and requires prolonged close or direct physical contact (e.g., kissing) for effective transmission. Asymptomatic carriage, present in <2% of children under 5 years of age and 20%-25% of adolescents and young adults, is a key component of natural transmission and maintenance of the pathogen, 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] Generally, the age group with the highest carriage rates is adolescents and young adults, who are more likely to engage in behaviors that are recognized risk factors for carriage and consequently, 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 can affect the incidence of IMD in other age groups, and it has been demonstrated in many European countries that vaccination campaigns with serogroup C conjugate vaccines have led to 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 severe illness caused by N. meningitidis (including N. meningitidis serogroup B), and symptoms include severe headache, fever, nausea, vomiting, photophobia, neck stiffness, lethargy, muscle pain, and a characteristic petechial rash (Harrison et al., [ed.] Orenstein WA, Offit PA, Edwards KM, Plotkin SA. Vaccines. 7. Philadelphia (PA): Elsevier; 2018. pp. 619-43). IMD can lead to meningococcal meningoencephalitis and meningococcemia. Meningococcemia is perhaps the most acutely fatal infectious disease in humans, with approximately 90% of deaths reported within the first two days of hospitalization. In 6% to 15% of patients with IMD, an inflammatory syndrome may develop due to the deposition of antigen-antibody complexes composed primarily of capsular polysaccharides, specific immunoglobulins, and complement fraction C3. These reactions generally occur 4 to 12 days after disease onset and include arthritis (mostly monoarticular (7% to 14% of patients)), cutaneous vasculitis, iritis, episcleritis, pleuritis, and pericarditis. Concomitant symptoms include recurrent fever, leukocytosis, and elevated serum C-reactive protein. Other complications that may occur in IMD patients include activated herpes simplex infection, symmetric distal necrosis, widespread ulcers in the histological distribution of vasculitis, gastrointestinal bleeding, subdural effusion, myocarditis, rhabdomyolysis, adult respiratory distress syndrome, acid-base and electrolyte disturbances, cerebral infarction, and intracranial suppuration.

[0009] Survivors of IMD may experience sequelae. The risk of neurological sequelae is 7%-12% (lower than that of pneumococcal meningitis), occurring primarily in infants. Hearing loss (permanent or transient) is the most common complication, occurring in approximately 4% of cases. Other sequelae include, among others, visual abnormalities, hydrocephalus, ataxia, aphasia, movement disorders, developmental delay, arthritis, spasticity, convulsions, renal failure, osteonecrosis, atrophic scarring, loss of parts of limbs, learning disabilities, and behavioral disorders (Batista et al., Asian Pac J Trop Med. 2017;10(11):1019-29; Stephens et al., Neisseria meningitidis. [ed.] J.E.Bennett, R. Dolin and M.J.Blaser. Philadelphia: Elsevier Saunders; 2015. p. 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 local epidemics and has been implicated in multiple prolonged outbreaks in several industrialized countries (Vuocolo et al., Hum Vaccin Immunother. 2018;14(5):1203-15), such as Cuba (Rodriguez et al., Mem Inst Oswaldo Cruz. 1999;94(4):433-40), Norway (Fredriksen et al., NIPH Ann. 1991;14(2):67-79;discussion-80), and New Zealand (Martin et al., J Infect Dis. 1998;177(2):497-500; Dyet et al., Epidemiol Infect. 2006;134(2):377-83). Smaller outbreaks caused by single strains have also been reported in other countries, e.g., 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 were 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] Recently, two vaccines targeting N. meningitidis serogroup B have been licensed: the four-component MenB protein vaccine (4CMenB), BEXSERO® from GlaxoSmithKline, Inc. [GSK], and the bivalent recombinant fHBP protein-based (rLP2086) vaccine (TRUMENBA® from Pfizer). These vaccines are adjuvanted with aluminum hydroxide and aluminum hydroxyphosphate adjuvants, respectively.

[0012] Adjuvants are agents incorporated into vaccine formulations to enhance the immunogenicity of vaccine antigens. Aluminum salts, such as aluminum phosphate and aluminum hydroxide, are the most commonly used vaccine adjuvants in humans and animals today. Although many aluminum-containing adjuvants are available, the adjuvant / antigen combination must be appropriately selected for any specific vaccine formulation to be immunogenic and stable for a long period of time.

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

[0014] Aluminum hydroxide, in its dehydrogenated crystalline form, is chemically aluminum oxyhydroxide [AlO(OH)], but in its aqueous phase, it acquires an additional water molecule to become aluminum trihydroxide [Al(OH)3] (Stanley L Hem, 2007). Aluminum oxyhydroxide has a PZC of 11, and therefore 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., proteins with acidic pIs). The PZC of aluminum hydroxide can be decreased by titration with phosphate ions. This is caused by ligand exchange between hydroxyl ions and phosphate ions on the surface of the aluminum hydroxide.

[0015] The adsorption of antigens to aluminum adjuvants depends on the physical and chemical properties of the antigen, the type of aluminum adjuvant used, and the adsorption conditions. Factors that can affect the adsorption of antigens to aluminum adjuvants include electrostatic forces, hydrophobic interactions, van der Waals forces, hydrogen bonds, pH, temperature, and the particle size of the adjuvant particles. Generally, antigens are adsorbed to aluminum adjuvants by electrostatic attraction (i.e., the adjuvant and the antigen have opposite charges) and / or by ligand exchange (i.e., phosphate groups on the antigen replace hydroxyl groups on the adjuvant surface) (Seeber SJ, 1991; Iyer S, 2004). The electrostatic interaction between aluminum adjuvants and antigen proteins can be affected by the pH of the formulation, the point of zero charge (PZC) of aluminum, and the isoelectric point (pI) of the protein. A properly balanced electrostatic interaction is usually required, as too strong a binding of the protein to aluminum can result in a poor induction of an immune response, while a weak interaction can result in poor adsorption and a low stability of the formulated product.

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

[0017] Adsorption can affect protein structure and stability. Research results on the effects of adsorption to aluminum-containing adjuvants are not all consistent. In one study, three proteins (bovine serum albumin [BSA], lysozyme, and ovalbumin) were destabilized after adsorption to Alhydrogel® or Adju-Phos®. In another study, adsorption to aluminum hydroxide stabilized the structure of BSA and β-lactoglobulin (BLG) (Jones, 2005; Zheng, 2007). Methods for stabilizing liquid vaccine formulations containing aluminum salt adjuvants include lyophilization, freezing, and freeze-drying, but these methods often result in adjuvant agglomeration, reduced immunogen concentration, and loss of immunogenicity (Maa, 2003; Diminsky, 1999; Alving, 1993; Warren, 1986). Even when such formulations are kept under refrigerated conditions (e.g., 2°C to 8°C), the adsorbed antigens may be chemically unstable and may undergo hydrolysis and fragmentation over time.

[0018] WO 2010 / 109323 discloses an immunogenic composition comprising a factor H binding protein (fHBP) antigen adsorbed to an aluminum hydroxyphosphate adjuvant. To ensure efficient adsorption of the antigen, the aluminum hydroxyphosphate adjuvant is selected to have a PZC in the range of 5.0 to 7.0. Furthermore, the pH of the composition is selected to be within 1.2 pH units of the PZC. Summary of the Invention [Problem to be solved by the invention]

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

[0020] There is a need to provide immunogenic compositions that include aluminum adjuvants and that are capable of inducing and enhancing immune responses.

[0021] There is a need to provide immunogenic compositions containing aluminum adjuvants that offer good safety profiles.

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

[0023] There is a need to provide immunogenic compositions capable of inducing an enhanced immune response to heterologous antigens, including combinations of Neisseria meningitidis serogroup B antigens.

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

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

[0026] There is a need to provide immunogenic compositions comprising combinations of Neisseria meningitidis serogroup B antigens that can improve the stability of the antigens in the composition.

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

[0028] The present disclosure aims to meet all or part of these needs. [Means for solving the problem]

[0029] According to one of its objects, the present disclosure relates to 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, and an aluminum hydroxyphosphate (AlPO4) adjuvant selected as having a point of 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, the combination comprising at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and an aluminum hydroxyphosphate (AlPO4) adjuvant, the aluminum hydroxyphosphate (AlPO4) adjuvant having a PZC of less than 5 prior to incorporation into the composition.

[0031] The composition of the present disclosure includes 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 an aluminum hydroxyphosphate (AlPO4) adjuvant. The AlPO4 adjuvant is selected to have a "point of zero charge" (PZC) below 5. fHBP A and B have an isoelectric point (pI) above the PZC of the adjuvant. The pH of the composition is at least 1.2 units higher than the PZC of the AlPO4 adjuvant. The composition has a pH of about 5.5 to about 7.0. About 50% to about 85% of the fHBP A and / or B is adsorbed to the AlPO4 adjuvant. The composition may further include at least one of NadA or dOMV antigens.

[0032] In the present disclosure, the AlPO4 adjuvant has a PZC below 5 prior to incorporation in an immunogenic composition.

[0033] Surprisingly, the inventors observed that by selecting an aluminum hydroxyphosphate (AlPO4) adjuvant with a PZC below 5 - contrary to the recommendations of WO 2010 / 109323 - for the formulation of an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens with fHBP antigen, it was possible to enhance the immune response to the antigen, increase the coverage of N. meningitidis group B strains and further stabilize the antigen.

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

[0035] Surprisingly, this beneficial effect was observed at fHBP antigen adsorption below 85%.

[0036] As shown in this Example section, selecting an AlPO4 adjuvant with a PZC below 5 for the preparation of an immunogenic composition comprising at least one factor H binding protein (fHBP) B enables the composition to induce an enhanced immune response (measured by antibody geometric mean titers - GMTs) against the fHBP B of the composition and the cognate fHBP B compared to compositions prepared with AlPO4 with a PZC above 5.

[0037] As shown in this Example section, selecting an AlPO adjuvant with a PZC below 5 for the preparation of an immunogenic composition comprising at least one factor H binding protein (fHBP) B also enables the composition to induce an enhanced immune response (measured by GMT and responder rate) against the composition's fHBP B and heterologous fHBP B compared to compositions prepared with AlPO having a PZC above 5.

[0038] As shown in this Example section, selecting an AlPO adjuvant with a PZC below 5 for the preparation of an immunogenic composition comprising at least one factor H binding protein (fHBP) A also enables the composition to induce an enhanced immune response (measured by GMT and responder rate) against the fHBP A antigen of the composition and its cognate fHBP A, compared to compositions prepared with AlPO having a PZC above 5.

[0039] As shown in this Example section, selecting an AlPO adjuvant with a PZC below 5 for the preparation of an immunogenic composition comprising at least one factor H binding protein (fHBP) A also enables the composition to induce an enhanced immune response (measured by GMT and responder rate) against the composition's fHBP A and a heterologous fHBP A compared to compositions prepared with AlPO having a PZC above 5.

[0040] An enhanced immune response is observed by an increase in the geometric mean titer (GMT) of functional serum bactericidal antibody activity (hSBA) using human complement achieved with the compositions of the present disclosure compared to the hSBA GMT achieved with a composition containing AlPO4 having a PZC greater than 5.

[0041] Furthermore, an enhanced immune response is observed by an increase in the % responder rate achieved with the compositions of the present disclosure compared to the % responder rate achieved with compositions containing AlPO4 having a PZC greater than 5.

[0042] An enhanced immune response can be observed by an increase in hSBA GMT fHBP-specific responses and / or an increase in the % responder rate.

[0043] Furthermore, as shown in this Example section, for the preparation of immunogenic compositions according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens including at least one fHBP A and / or at least one fHBP B, selecting an AlPO adjuvant with a PZC below 5 advantageously does not compromise the stability of the fHBP.

[0044] As shown in this Example section, for the preparation of immunogenic compositions according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens with at least one fHBP A, selecting an AlPO adjuvant with a PZC below 5 can enhance the stability of fHBP A compared to compositions prepared with an AlPO adjuvant with a PZC above 5.

[0045] The AlPO4 adjuvant may be selected to have a PZC in the range of about 4.1 to less than 5, or in the range of about 4.2 to about 4.9, or in the range of about 4.3 to about 4.8, or about 4.5.

[0046] The AlPO4 adjuvant may be selected to have a PZC of about 4.5.

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

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

[0049] The immunogenic composition may have a pH in the range of about 5.5 to about 7.0, or may have a pH of 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 Neisserial adhesin A (NadA) protein.

[0051] As shown in this Example section, for the preparation of immunogenic compositions according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens with at least one NadA protein, selecting an AlPO4 adjuvant with a PZC below 5 can enhance the stability of the NadA protein compared to compositions prepared with AlPO4 having a PZC above 5.

[0052] As shown in this Example section, for the preparation of immunogenic compositions according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens including fHBP A and NadA proteins, selecting an AlPO adjuvant with a PZC below 5 can enhance the stability of the fHBP A and NadA proteins compared to compositions prepared with AlPO having a PZC above 5.

[0053] Furthermore, as shown in this Example section, for the preparation of immunogenic compositions according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens with at least one NadA protein, selecting an AlPO4 adjuvant with a PZC below 5 advantageously does not impair the immune response induced by the NadA protein.

[0054] Furthermore, as shown in this Example section, for the preparation of immunogenic compositions according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens with at least one detergent-extracted outer membrane vesicle (dOMV), selecting an AlPO4 adjuvant with a PZC below 5 advantageously does not impair the immune response or stability induced by the dOMV.

[0055] Furthermore, as shown in this Example section, for the preparation of immunogenic compositions according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens with at least one detergent-extracted outer membrane vesicle (dOMV), selecting an AlPO4 adjuvant with a PZC below 5 advantageously does not increase the pyrogenicity of the composition.

[0056] Furthermore, as shown in this Example section, for the preparation of 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, selecting an AlPO4 adjuvant with a PZC below 5 advantageously does not increase the pyrogenicity of the composition.

[0057] In the immunogenic compositions of the present disclosure, fHBP B may be adsorbed to an AlPO adjuvant in an amount of about 85% or less of the total amount of fHBP B present in the composition, or in an amount ranging from about 50% to less than 85% of the total amount of fHBP B present in the composition.

[0058] fHBP B may have an isoelectric point (pI) above the PZC of the AlPO4 adjuvant.

[0059] fHBP B may have an isoelectric point (pI) in the range of about 5.0 to about 7.0, or 5.2 to about 6.5, or about 5.3 to about 6.0, or may be about 5.5 or 5.46.

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

[0061] fHBP B can be non-lipidated.

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

[0063] The fHBP B can be a mutant fHBP B that comprises 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 comprises or consists 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, fHBP A may be adsorbed to the AlPO adjuvant in an amount of about 85% or less of the total amount of fHBP A present in the composition, or in an amount ranging from about 50% to less than 85% of the total amount of fHBP A present in the composition.

[0067] fHBP A may have an isoelectric point (pI) ranging 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 5.9 or 5.86.

[0068] The fHBP A may be non-lipidated.

[0069] The fHBP A can be a mutant fHBP A that includes at least one mutation that reduces or prevents binding of fHBP A to human factor H (fH).

[0070] The fHBP A can be a mutant protein 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: 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, fHBP A and / or fHBP B may each 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, for example, from about 0.2 mL to about 0.8 mL, from about 0.4 mL to about 0.6 mL, or may be about 0.5 mL.

[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 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 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 at about 50 μg / dose.

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

[0080] The Porin A (PorA) protein may be selected from the PorA VR2 subtype, or is PorA VR2 P1.2.

[0081] The dOMV may be present in an amount ranging from about 5 μg / dose to about 400 μg / dose, or from about 10 μg / dose to about 300 μg / dose, or from about 25 μg / dose to about 250 μg / dose, or from about 35 μg / dose to about 225 μg / dose, or from about 50 μg / dose to about 200 μg / dose, or from about 75 μg / dose to about 180 μg / dose, or from about 100 μg / dose to about 150 μg / dose, or from about 110 μg / dose to about 125 μg / dose, or at 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 among 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 compositions of the present disclosure may comprise or consist of 25-100 μg / dose of non-lipidated fHBP A consisting of SEQ ID NO: 2, 25-100 μg / dose of non-lipidated fHBP B consisting of SEQ ID NO: 4, 25-100 μg / dose of NadA protein consisting of SEQ ID NO: 5, 20-250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, 100-800 μg / dose of AlPO adjuvant at a PZC of about 4.5, 50 mM acetate buffer, pH 6.0.

[0086] The immunogenic compositions of the present disclosure may comprise or consist of 25-100 μg / dose of non-lipidated fHBP A consisting of SEQ ID NO: 8, 25-100 μg / dose of non-lipidated fHBP B consisting of SEQ ID NO: 9, 25-100 μg / dose of NadA protein consisting of SEQ ID NO: 5, 20-250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, 100-800 μg / dose of an AlPO4 adjuvant at a PZC of about 4.5, 50 mM acetate buffer, pH 6.0.

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

[0088] The conjugated capsular saccharide may be conjugated to a tetanus toxoid carrier.

[0089] As shown in this Examples section, for the preparation of immunogenic compositions according to the present disclosure comprising a combination of N. 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, selecting an AlPO4 adjuvant with a PZC below 5 advantageously does not compromise the stability of the conjugated capsular saccharide or the immune response induced thereby.

[0090] The immunogenic compositions of the present disclosure have a sedimentation onset time (T) ranging from about 3.5 minutes to about 10 minutes. onset ).

[0091] The settling onset time can be measured by using static multiple light scattering to detect particle movement and size fluctuations in the dispersion, as disclosed in the Examples section. Two detection lights can be used—transmission and backscattering—and the signal is related to particle size and concentration, the fluctuations of which indicate the occurrence of destabilization. This parameter is a measure of the physical stability of the composition and is related to the flocculation properties of the suspension. Other methods known in the art can also be used to determine the settling onset time.

[0092] Advantageously, a settling onset time of at least 3.5 minutes or greater ensures that the components of the composition remain in suspension during manufacturing operations, thus allowing for better manufacturing control.

[0093] The immunogenic compositions of the present disclosure may enhance immune responses against N. meningitidis serogroup B strains that express a fHBP B heterologous to the fHBP B of the composition.

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

[0095] The immunogenic compositions of the present disclosure may enhance immune responses against N. meningitidis serogroup B strains that express a fHBP A heterologous to the fHBP A of the composition.

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

[0097] The immunogenic compositions of the present disclosure may enhance the stabilization of fHBP A.

[0098] The immunogenic compositions of the present disclosure may enhance the stabilization 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 for use in methods of inducing an immune response against Neisseria meningitidis group B strains.

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

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

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

[0104] According to another of its objects, the present disclosure relates to the use of an AlPO adjuvant having a PZC of less than 5 to enhance the immune response induced by a composition comprising a N. meningitidis fHBP A antigen against a N. meningitidis serogroup B strain expressing an fHBP A homologous to the 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 below 5 to stabilize 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 below 5 to stabilize at least one Neisserial adhesin A (NadA) protein in an immunogenic composition.

[0107] According to another of its objects, the present disclosure provides a method for determining the sedimentation time (T) of a 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. onset The present invention relates to the use of an AlPO4 adjuvant having a PZC of less than 5 to stabilize a soluble ...

[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 to adjuvant 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 to prepare 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.

[0110] According to another of its objects, the present disclosure provides a method for producing 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 one factor H binding protein (fHBP) B, and an AlPO4 adjuvant, the method comprising at least: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AIPO4 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 combinations being carried out in any order; The present invention relates to a method comprising:

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

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

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

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

[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 provides a method for preparing an immunogenic composition comprising a N. meningitidis fHBP A antigen, the method inducing an enhanced immune response against a N. meningitidis serogroup B strain expressing a fHBP A heterologous to the fHBP A antigen of the composition, the method comprising at least: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AlPO4 adjuvant selected in step a) with the fHBP A antigen; c) obtaining said immunogenic composition; The present invention relates to a method comprising:

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

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

[0120] 1. A method of enhancing an immune response induced by a composition comprising a N. meningitidis fHBP B antigen in an individual in need thereof against a N. meningitidis serogroup B strain expressing an fHBP B heterologous to the fHBP B antigen of the composition, the method comprising at least the step of administering to the individual an immunogenic composition or vaccine according to the present disclosure, wherein said administering step induces an enhanced immune response against the N. meningitidis serogroup B strain expressing the heterologous fHBP B.

[0121] 1. A method of enhancing an immune response induced by a composition comprising a N. meningitidis fHBP B antigen in an individual in need thereof against a N. meningitidis serogroup B strain expressing an fHBP B homologous to the fHBP B antigen of the composition, the method comprising at least the step 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 N. meningitidis serogroup B strain expressing the homologous fHBP B.

[0122] 1. A method of enhancing an immune response induced by a composition comprising a N. meningitidis fHBP A antigen in an individual in need thereof against a N. meningitidis serogroup B strain expressing an fHBP A heterologous to the fHBP A antigen of the composition, the method comprising at least the step of administering to the individual an immunogenic composition or vaccine according to the present disclosure, wherein said administering step induces an enhanced immune response against the N. meningitidis serogroup B strain expressing the heterologous fHBP A.

[0123] 1. A method of enhancing an immune response induced by a composition comprising a N. meningitidis fHBP A antigen in an individual in need thereof against a N. meningitidis serogroup B strain expressing an fHBP A homologous to the fHBP A antigen of the composition, the method comprising at least the step 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 N. meningitidis serogroup B strain expressing the homologous fHBP A. [Brief explanation of the drawings]

[0124] [Figure 1]1 shows the results of hSBA assays against a Neisseria meningitidis strain expressing fHBP A56, which is closely related (also called homologous) to A05, in sera (purified IgG) collected at D0 (gray) 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 (PZC4.5). [Figure 2] Figure 1 shows the results of hSBA assays against a Neisseria meningitidis strain expressing the closely related (homologous) fHBP B44 to B01 in sera (purified IgG) collected at D0 (gray) 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 (PZC4.5). [Figure 3] 1 shows the results of hSBA assays against a Neisseria meningitidis strain expressing A05 and heterologous fHBP A22 in sera (purified IgG) collected at D0 (gray) 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 (PZC4.5). [Figure 4] 1 shows the results of serum (purified IgG) collected at D0 (gray) 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 (PZC4.5) measured against a Neisseria meningitidis strain expressing B01 and heterologous fHBP B24. [Figure 5]1 shows the results of hSBA assays against a Neisseria meningitidis strain expressing VR2-P1.2-PorA in sera (purified IgG) collected on D0 (gray) and D42 (black) from rabbits immunized on D0 and D28 with a MenB immunogenic composition formulated with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC4.5). [Figure 6] 1 shows the results of hSBA assays against a NadA-expressing Neisseria meningitidis strain in sera (purified IgG) collected on D0 (gray) and D42 (black) from rabbits immunized on D0 and D28 with a MenB immunogenic composition formulated with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC4.5). [Figure 7] Figure 1 shows the results of hSBA assays against Neisseria meningitidis strain A in sera (purified IgG) collected at DO, D28, and D42 from rabbits immunized at DO and D28 with MenACWY immunogenic composition formulated without AlPO4 adjuvant (white), with mod-AlPO4 adjuvant (PZC4.5) (shaded), or with MenB antigen and mod-AlPO4 adjuvant (PZC4.5) (black). [Figure 8] Figure 1 shows the results of hSBA assays against Neisseria meningitidis strain C in sera (purified IgG) collected at DO, D28, and D42 from rabbits immunized at DO and D28 with MenACWY immunogenic composition formulated without AlPO4 adjuvant (white), with mod-AlPO4 adjuvant (PZC4.5) (shaded), or with MenB antigen and mod-AlPO4 adjuvant (PZC4.5) (black). [Figure 9]Figure 1 shows the results of hSBA assays against Neisseria meningitidis strain W135 in sera (purified IgG) collected on DO, D28, and D42 from rabbits immunized on DO and D28 with MenACWY immunogenic composition formulated without AlPO4 adjuvant (white), with mod-AlPO4 adjuvant (PZC4.5) (shaded), or with MenB antigen and mod-AlPO4 adjuvant (PZC4.5) (black). [Figure 10] Figure 1 shows the results of hSBA assays against Neisseria meningitidis strain Y in sera (purified IgG) collected at DO, D28, and D42 from rabbits immunized at DO and D28 with MenACWY immunogenic compositions formulated without AlPO4 adjuvant (white), with mod-AlPO4 adjuvant (PZC4.5) (shaded), or with MenB antigen and mod-AlPO4 adjuvant (PZC4.5) (black). [Figures 11A-11C] Figure 1 shows the results of the relative antigenicity (RA) of A05tmN, B01smN, NadA and dOMV formulated into MenPenta immunogenic compositions with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC4.5) and subjected to heat stress at 45°C (or 37°C for NadA) for 20 days. [Figures 12A-12D] Figure 1 shows the percent change in free polysaccharide for serogroups A, C, W-135, and Y formulated into MenPenta immunogenic compositions with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC4.5) and subjected to heat stress at 5°C or 45°C for 28 days. [Figures 13A-13D] The stability of A05tmN, B01smN, NadA and dOMV in AlPO4 adjuvants of different points of zero charge (PZC), AlPO4 adjuvants (PZC 5.2) or mod-AlPO4 adjuvants (PZC 4.3, 4.5 or 4.8) over a 30 day period at 45°C for B01, A05 and dOMV or 37°C for NadA is shown, expressed as the relative antigenicity (RA) of A05tmN, B01smN, NadA or dOMV. DETAILED DESCRIPTION OF THE INVENTION

[0125] Array Description SEQ ID NO: 1 represents the wild-type sequence of fHBP A05 lacking the first 19 amino acids from the N-terminus, which correspond to the signal peptide involved in lipid addition. [ka]

[0126] SEQ ID NO: 2 represents the mutated fHBP A05 sequence lacking the signal peptide involved in lipidation and having the mutations G220S, L130R, G133D (numbering determined with reference to the sequence of SEQ ID NO: 6 (fHBP B24)). [ka]

[0127] SEQ ID NO: 3 represents the fHBP B01 wild-type sequence without the signal peptide involved in lipidation. [ka]

[0128] SEQ ID NO: 4 represents the mutated fHBP B01 sequence lacking the signal peptide involved in lipidation and having the mutation H248L (numbering is determined with reference to the sequence of SEQ ID NO: 6 (fHBP B24)). [ka]

[0129] SEQ ID NO: 5 represents the NadA1 sequence from MenB strain MC58, lacking 23 amino acids of the signal peptide at the N-terminus and the last 55 amino acids at the C-terminus. [ka]

[0130] SEQ ID NO: 6 represents the fHBP B24 wild-type sequence from which the numbering of the mutation positions in A05 and B01 is determined. [ka]

[0131] SEQ ID NO: 7 represents the wild-type NadA1 sequence from MenB strain MC58. [ka]

[0132] SEQ ID NO: 8 represents the non-lipidated mutant fHBP A05 sequence having the mutations G220S, L130R, G133D (numbering determined with reference to the sequence of SEQ ID NO: 6 (fHBP B24)) and a substitution of the N-terminal cysteine with a methionine by fusing the ATG start codon directly to the second 5' codon in the DNA sequence encoding the recombinant protein. [ka]

[0133] SEQ ID NO: 9 represents the non-lipidated mutant fHBP B01 sequence having the mutation H248L (numbering is determined with reference to the sequence of SEQ ID NO: 6 (fHBP B24)) and a substitution of the N-terminal cysteine with a methionine by fusing the ATG start codon directly to the second 5' codon in the DNA sequence encoding the recombinant protein. [ka]

[0134] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press can provide those skilled in the art with a general dictionary of many of the terms used in this disclosure. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention. In the event of any conflict, the present specification, including definitions, will control. Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and medicinal chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Methods are performed according to kit manufacturer's specifications, as commonly accomplished in the art, or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0135] Units, prefixes, and symbols are expressed in their International System of Units (SI) accepted format. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino (N-) to carboxyl (-C) orientation. The headings provided herein are not limitations of the various aspects of this disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0136] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, such citation does not constitute an admission that any of those documents form part of the common general knowledge in the art.

[0137] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an antigen" includes a plurality of such antigens, a reference to "the protein" includes one or more proteins, etc.

[0138] It is understood that aspects and embodiments of the present disclosure described herein include "having," "including," "consisting of," and "consisting essentially of" aspects and embodiments. The words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," will be understood to imply the inclusion of one or more specified 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 one or more specified elements but the exclusion of any additional elements. The term "consisting essentially of" implies the inclusion of the specified elements, and possibly one or more other elements, if the other element or elements do not materially affect the basic and novel characteristic(s) of the disclosure. It is understood that variations of the present disclosure using the term "comprising" or equivalents encompass embodiments where that term is replaced with "consisting of" or "consisting essentially of."

[0139] Furthermore, "and / or," when used herein, should be interpreted as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or," when used herein in phrases such as "A and / or B," is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and / or," when used in phrases such as "A, B and / or C," is intended to encompass each of the following embodiments: 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).

[0140] The terms "approximately" or "about" are used herein to mean approximately, roughly, in the region of, or within a range of. When the term "about" is used in conjunction with a range of numerical values, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value by a variance, for example, of 10 percent upward or downward (high or low), above and below the stated value. In some embodiments, the term indicates 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 indicated numerical value. In some embodiments, "about" indicates a deviation of ±10% from the indicated numerical value. In some embodiments, "about" refers to a ±5% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±4% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±3% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±2% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±1% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the indicated numerical value.

[0141] Within the scope of this disclosure, the term "significantly" as used in connection with a change is intended to mean that the change observed is discernible and / or that it is statistically significant.

[0142] Within the scope of this disclosure, the term "substantially" when used in conjunction with a feature of the disclosure is intended to define a set of embodiments relating to that feature that are largely, but not entirely, similar to that feature.

[0143] Within the scope of the present disclosure, an "immunogenic composition" is intended to refer to a composition comprising at least one antigen in an amount and in a suitable formulation sufficient to induce an immune response directed against said antigen in an individual to whom the composition is administered. The immune response may be a humoral response and / or a cellular response.

[0144] Within the scope of 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 equal to zero.

[0145] Within the scope of this disclosure, "pI" is intended to mean the isoelectric point and is intended to refer to the pH at which a particular molecule carries no net electrical charge.

[0146] Within the scope of the present disclosure, the term "antigen" includes any molecule, e.g., a peptide or protein, that elicits an immune response and / or contains at least one epitope against which an immune response is directed. For example, an antigen is a molecule that, optionally after processing, induces an immune response specific to the antigen or a cell expressing the antigen. After processing, the antigen can be presented by an MHC molecule and react specifically with 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 be derived from a naturally occurring antigen.

[0147] Within the scope of the present disclosure, the term "adjuvant" is intended to refer to a compound capable of enhancing the immune response directed against an antigen, including enhancing the magnitude and / or duration of the immune response generated by the antigen.

[0148] Within the scope of this disclosure, the term "buffer" is intended to refer to an aqueous solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH. Buffers are used to maintain a stable pH in a solution because they can neutralize small amounts of additional acid or base. Suitable buffers for immunogenic compositions are known in the art.

[0149] As used 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, i.e., bacteria, viruses, and substances that appear foreign and harmful. The immune response may have a humoral component, i.e., antibodies, or a cellular component.

[0150] As used herein, the phrase "enhancing an immune response" is intended to refer to an immune response induced by a first immunogenic composition, measured in its humoral and / or cellular components, being greater than an immune response induced by a second immunogenic composition, similarly measured, where the first and second compositions differ in one parameter. This difference may be a trend or statistically significant. Within the scope of the present disclosure, to determine an enhanced immune response, a composition of the present disclosure is compared to a composition containing an AlPO4 adjuvant different from the AlPO4 of the present disclosure, where the other parameters of the compositions are otherwise identical.

[0151] As used herein, the terms "stabilize," "stabilizing," or "stabilization" in reference to an antigen are intended to refer to maintaining the immunogenic properties of the antigen at a given or stable level over a period of time. The stabilizing effect of a formulation in relation to an antigen can typically be demonstrated by reduced or no loss of immunogenicity or potency of the antigen during stress—physical, chemical, or mechanical (e.g., pH shift, temperature change, surface interaction, external impurities, mixing, etc.)—in the presence of the formulation, compared to the loss of immunogenicity of the antigen in the absence of the formulation or in the presence of another formulation. Immunogenicity or potency can be measured at a single time point or repeatedly over a period of time, e.g., 2, 3, 4, 6, or 8 time points over a 3, 6, 9, or 12-month period. The immunogenicity (or potency) of an antigen is its ability to induce an immune response and can be measured by any method known in the art.

[0152] Instability of protein antigens can be caused by chemical degradation or aggregation of molecules to form higher order polymers, by dissociation of heterodimers into monomers, by deglycosylation, modified glycosylation, or any other structural modification that reduces at least one biological activity of the antigen.

[0153] As used herein, the term "vaccine" is intended to mean an immunogenic composition directed against a pathogenic agent, which is administered to a subject to induce an immune response with the intent of protecting the subject from (i.e., conferring protective immunity to) or treating a disease caused by the pathogenic agent. A vaccine as disclosed herein can be used as a protective (prophylactic) vaccine administered to a subject prior to infection with the intent of preventing or reducing the likelihood of initial (and / or repeat) infection.

[0154] In the context of the present disclosure, the term "protective immunity" means that administration of a vaccine or immunization schedule to a mammal induces an immune response that prevents, delays the onset of, or reduces the severity of, disease caused by Neisseria meningitidis, or diminishes or eliminates disease symptoms altogether. 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 recognized in the art as a predictor of the protective efficacy of a vaccine in humans (Goldschneider et al. (1969) J. Exp. Med. 129:1307).

[0155] An "isolated" protein, or fragment, variant, or derivative thereof, refers to a protein that is not in its natural environment. No particular level of purification is required. For example, an isolated protein can simply be removed from its native or natural environment. A recombinantly produced protein expressed in a host cell is considered isolated for the purposes of this disclosure, as are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0156] 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., cows, 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.

[0157] It will be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications mentioned herein are incorporated by reference to disclose and describe such methods and / or materials in the context in which the publications are cited.

[0159] The list of suppliers, raw materials and ingredients as set forth below are listed as such, as are combinations and mixtures thereof, which are contemplated and are within the scope of this specification.

[0160] It should be understood that every upper limit of any numerical limitation provided throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every lower limit of any numerical limitation provided throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range provided throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0161] All lists of items, such as, for example, lists of ingredients, are intended to be Markush groups and should be construed as such. Thus, all lists may be read and construed as items "selected from the group consisting of" a list of items and combinations and mixtures thereof.

[0162] Trade names may be referenced herein for ingredients, including various raw materials, utilized in this disclosure. The inventors do not intend to be limited herein to any particular trade name materials. Equivalent materials (e.g., those available from different suppliers under different names or reference numbers) to those referenced by trade name may be substituted and utilized in the description herein.

[0163] Aluminum hydroxyphosphate (AlPO4) adjuvant The compositions of the present disclosure include an aluminum hydroxyphosphate adjuvant.

[0164] Aluminum hydroxyphosphate adjuvants, represented by the chemical formula Al(OH)PO4 and referred to herein as AlPO4 adjuvants, are not stoichiometric compounds; the amounts of hydroxyl and phosphate moieties depend on the preparation conditions. The respective ratios of hydroxyl and phosphate moieties affect the point of zero charge (PZC) of the adjuvant.

[0165] The PZC corresponds to the pH at which the surface has no net charge. The PZC is inversely proportional to the degree of substitution of phosphate for hydroxyl (P / Al molar ratio). As hydroxyl anions are replaced by phosphate anions, the PZC decreases. The PZC can be altered by changing the concentration of free phosphate ions in the solution (more phosphate = more acidic, i.e., lower PZC) or by adding a buffer, such as a histidine buffer (which makes the PZC more basic, i.e., higher).

[0166] AlPO4 adjuvants for use within the scope of this disclosure are selected as having a PZC of less than 5. The PZC is measured prior to introducing the AlPO4 adjuvant into the composition. Accordingly, AlPO4 adjuvants suitable for this disclosure are selected based on their PZC of less than 5 prior to their introduction into the immunogenic composition. The selected AlPO4 will therefore have a PZC of less than 5 prior to their introduction into the composition.

[0167] The AlPO4 adjuvant can be selected to have a PZC in the range of about 4.1 to less than 5, or in the range of about 4.2 to about 4.9, or in the range of about 4.3 to about 4.8, or in the range of about 4.4 to about 4.6, or about 4.5.

[0168] 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.

[0169] The AlPO4 adjuvant may be selected to have a PZC of about 4.3, 4.5, or 4.8.

[0170] The AlPO4 adjuvant may be selected to have a PZC of about 4.5.

[0171] The AlPO4 adjuvant may be selected to have a PZC of 4.5.

[0172] A variety of different methods can be used to obtain AlPO4 adjuvants in the target PZC.

[0173] To prepare an AlPO adjuvant with a target PZC, aluminum hydroxide (Al(OH)) or aluminum hydroxyphosphate adjuvants with a PZC higher than the target PZC can be used. Such AlPO adjuvants are commercially available.

[0174] In one method, a phosphate buffer pH 5.8 formulated with a combination of 0.5 M monobasic sodium phosphate and 0.5 M dibasic sodium phosphate can be added to aluminum hydroxide or to a higher PZC aluminum hydroxyphosphate adjuvant to create an AlPO adjuvant with a target PZC.

[0175] Alternatively, the PZC of an AlPO4 adjuvant can be modified by titrating a higher PZC AlPO4 adjuvant with a 0.5M monobasic sodium phosphate stock solution.

[0176] AlPO4 adjuvants can be prepared by batch precipitation with three reactants: aluminum chloride (or other aluminum source), sodium trisodium phosphate, and sodium hydroxide.

[0177] The P / Al molar ratio of an aluminum hydroxyphosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2 or between 0.85 and 1.0, more preferably about 0.9. A P / Al molar ratio of at least 0.5 may confer better immunogenicity and stability properties to the adjuvant.

[0178] AlPO4 adjuvants are generally amorphous (i.e., X-ray amorphous). They are generally particulate (e.g., they exhibit a platelet-like morphology when viewed under a transmission electron microscope). Typical diameters of the plates can be 10-100 nm, and they form aggregates of 0.5-20 μm (e.g., about 1-10 μm) in size. For aluminum hydroxyphosphate adjuvants, 1 mg of AlPO4 at pH 7.4 3+ The adsorption capacity of 0.7 to 1.5 mg of protein per 1000g of silica has been reported.

[0179] A typical adjuvant is amorphous aluminum hydroxyphosphate with a P / Al molar ratio of 0.84-0.92; this adjuvant contains 0.8 mg Al 3+ / mL.

[0180] Al(Al 3+ The concentration of Al) may preferably be less than 5 mg / mL, e.g., <4 mg / mL, <3 mg / mL, <2 mg / mL, <1 mg / mL, etc. Suitable ranges may be from 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.

[0181] The aluminum phosphate adjuvant may be present in the compositions disclosed herein in an amount ranging from about 100 μg / dose to about 1000 μg / dose, or from about 150 μg / dose to about 900 μg / dose, or from about 200 μg / dose to about 800 μg / dose, or from about 250 μg / dose to about 700 μg / dose, or from about 300 μg / dose to about 600 μg / dose, or from about 350 μg / dose to about 550 μg / dose, or from about 400 μg / dose to about 500 μg / dose, or from about 400 μg / dose to about 800 μg / dose, or about 400 μg / dose, or about 800 μg / dose.

[0182] The aluminum phosphate adjuvant may be present in the compositions disclosed herein in an amount of about 400 μg / dose.

[0183] In the compositions of the present disclosure, fHBP can be adsorbed to an AlPO4 adjuvant in an amount of about 85% or less of the total amount of fHBP in the composition, or in an amount ranging from about 50% to less than 85%, or from about 70% to about 80% of the total amount of fHBP in the composition. fHBP can be adsorbed to an AlPO4 adjuvant in an amount ranging from about 50% to about 85%, or less than 85%, or from about 50 to about 80%, or from about 50 to about 75%, or from about 65 to about 75% of the total amount of fHBP in the composition.

[0184] In the immunogenic compositions of the present disclosure, fHBP B may be adsorbed to an AlPO4 adjuvant in an amount of about 85% or less of the total amount of fHBP B present in the composition, or in an amount ranging from about 50% to less than 85% of the total amount of fHBP B present in the composition. fHBP B may be adsorbed to an AlPO4 adjuvant in an amount ranging from about 50% to about 85%, or less than 85%, or from about 50% to about 80%, or from about 50% to about 75%, or from about 65% to about 75% of the total amount of fHBP B in the composition. fHBP B may be adsorbed to an AlPO4 adjuvant in an amount of about 50, 55, 60, 65, 70, 75, or 80%, or less than 85% of the total amount of fHBP B present in the composition.

[0185] In the immunogenic compositions of the present disclosure, fHBP A may be adsorbed to an AlPO4 adjuvant in an amount of about 85% or less of the total amount of fHBP A present in the composition, or in an amount ranging from about 50% to less than 85% of the total amount of fHBP A present in the composition. fHBP A may be adsorbed to an AlPO4 adjuvant in an amount ranging from about 50% to about 85%, or less than 85%, or from about 50% to about 80%, or from about 50% to about 75%, or from about 65% to about 75% of the total amount of fHBP A in the composition. fHBP A may be adsorbed to an AlPO4 adjuvant in an amount of about 50, 55, 60, 65, 70, 75, 80%, or less than 85% of the total amount of fHBP A present in the composition.

[0186] The proportion of fHBP adsorbed can be controlled by varying the salt concentration and / or pH during formulation; for example, higher NaCl concentrations generally result in lower fHBP adsorption to AlPO adjuvants. The amount of adsorption for any given formulation will depend on a combination of parameters, including the PZC of the adjuvant, the salt concentration and pH during formulation, the adjuvant concentration, the antigen concentration, and the pI of the antigen. The effect of each of these parameters on adsorption can be readily determined. The degree of adsorption can be determined by comparing the total amount of fHBP antigen in the composition (e.g., measured before adsorption occurs or by desorbing the adsorbed antigen) with the amount remaining in the supernatant after centrifugation. A suitable method may be that disclosed in the Examples section.

[0187] In some embodiments, the AlPO adjuvants of the present disclosure are used in compositions having a pH ranging from about 5.5 to about 7.0. The compositions of the present disclosure may have a pH of about 6.0.

[0188] In some embodiments, the AlPO4 adjuvants of the present disclosure are used in compositions having a pH such that the difference between the PZC of the AlPO4 adjuvant and the pH of the composition ranges from about 0.6 to about 2.9.

[0189] The composition may have a pH of 0.6 to 2.9 units from the PZC of the AlPO4 adjuvant, or 1.0 to 2.8, or 1.2 to 2.5, or 1.4 to 2.1 units from the PZC of the adjuvant.

[0190] The composition may have a pH that is 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 from the PZC of the AlPO4 adjuvant.

[0191] In some embodiments, the AlPO4 adjuvants of the present disclosure are used in compositions having a pH such that the difference between the PZC of the AlPO4 adjuvant and the pH of the composition ranges from about 1.0 to about 2.9, or from about 1.2 to about 2.9.

[0192] The composition may have a pH that is at least 1.2 units from the PZC of the AlPO4 adjuvant.

[0193] The composition may have a pH that is 2.9 units or less from the PZC of the AlPO4 adjuvant.

[0194] The composition may have a pH that is 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 from the PZC of the AlPO4 adjuvant.

[0195] The composition may have a pH that is 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 from the PZC of the AlPO4 adjuvant.

[0196] The composition may have a pH that is 1.2, 1.5, 1.7, 2.2, 2.5 or 2.7 units from the PZC of the AlPO4 adjuvant.

[0197] The composition may have a pH that is 1.2, 1.3, 1.4, 1.5, 1.6 or 1.7 units from the PZC of the AlPO4 adjuvant.

[0198] The composition may have a pH that is 1.2, 1.5, or 1.7 units from the PZC of the AlPO4 adjuvant.

[0199] The composition may have a pH of about 5.5 to about 7.0. The composition may have a pH of 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.

[0200] The composition may have a pH of about 5.5, 6.0, 6.5 or about 7.0.

[0201] The composition may have a pH of about 6.0.

[0202] An AlPO4 adjuvant may have a PZC of about 4.5.

[0203] In some embodiments, the AlPO4 adjuvants of the present disclosure are used with fHBP having an isoelectric point (pI) in the range of about 5 to about 7.

[0204] In some embodiments, the difference between the PZC of the AlPO4 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.

[0205] In some embodiments, the difference between the PZC of the AlPO4 adjuvant and the pI of the fHBP antigen may be about 0.96 or about 1.66.

[0206] The isoelectric point of fHBP antigen can be experimentally determined by techniques such as isoelectric focusing. However, more conveniently, the isoelectric point is the theoretical isoelectric point. This can be calculated using the pKa values of amino acids described by Bjellqvist et al. ((1993) Electrophoresis 14:1023-31) and the associated ExPASy tool (Gasteiger et al. (2005) Protein Identification and Analysis Tools on the ExPASy Server in The Proteomics Protocols Handbook (ed. John M. Walker), Humana Press (2005)).

[0207] antigen The immunogenic compositions disclosed herein include a combination of Neisseria meningitidis serogroup B antigens, including at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and an aluminum hydroxyphosphate (AlPO4) adjuvant, the AlPO4 adjuvant having a point of zero charge (PZC) of less than 5.

[0208] fHBP 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 nearly all invasive meningococcal isolates. fHBP is an important virulence factor because it binds to human complement factor H (fH), a negative regulator of the alternative complement pathway (Seib et al., Expert Rev Vaccines. 2015;14(6):841-59). Binding of fHBP to human fH allows the pathogen to evade killing via the alternative complement pathway by the host innate immune system and survive in human serum and blood.

[0209] Three major genetic and immunological fHBP variants have been described: variant 1, which corresponds to subgroup B, and variants 2 and 3, both of which are classified as subgroup A (Seib et al., Expert Rev Vaccines. 2015;14(6):841-59). In addition to the nomenclature provided by Pfizer (fHBP A and B) and Novartis (variants 1, 2, and 3), fHBPs are identified by unique ID numbers in the PubMLST database. Although there is significant antigenic variability between fHBP subgroups A and B, the protein sequences within each subgroup are highly conserved among different strains (greater than 86% sequence identity). According to the neisseria.org or pubmlst.org / neisseria / fHBP / website, each unique fHBP found in N. meningitidis is also assigned an fHBP peptide ID. Because the length of the variant 2 (v.2) fHBP protein (from strain 8047, fHBP ID 77) and variant 3 (v.3) fHBP (from strain M1239, fHBP ID 28) differs by −1 and +7 amino acid residues, respectively, compared to MC58 (fHBP ID 1, which is selected as the reference sequence for numbering), the numbering used to refer to residues in the v.2 and v.3 fHBP proteins differs from the numbering based on the actual amino acid sequences of these proteins. Thus, for example, when referring to a leucine residue (L) at position 166 of the v.2 or v.3 fHBP sequence, it refers to the residue at position 165 in the v.2 protein and the residue at position 173 in the v.3 protein. Members of variants 1, 2, and 3 are present in approximately 65%, 25%, and 10%, respectively, of MenB clinical isolates causing invasive disease.In the United States and Europe combined, the 10 most prevalent fHBP variants occurring in the global population of MenB strains account for approximately 80% of invasive disease-causing strains (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).

[0210] A fHBP in this disclosure can be a wild-type (naturally occurring) polypeptide or can be non-naturally occurring (modified by amino acid substitution, insertion or deletion), provided that the polypeptide is capable of eliciting an immune response.

[0211] The fHBP to be used according to the present disclosure may be 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 stage of the protein. The lipidation signal peptide is specific to each type of protein and to the host cell in which the protein is produced.

[0212] The fHBP polypeptide is expressed in N. meningitidis as a precursor protein with a lipoprotein signal motif at its N-terminus. During processing, this motif is cleaved, leaving an N-terminal cysteine residue that, when cotranslationally modified with a lipid anchor, anchors the protein to the Neisserial outer membrane (McNeil et al. (2013) MMBR 77(2):234-252). For lipidated fHBP, the lipid attached to the cysteine typically contains a palmitoyl residue, such as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys), dipalmitoyl-S-glycerylcysteine (Pam2Cys), or N-acetyl (dipalmitoyl-S-glycerylcysteine).

[0213] To avoid lipidation of recombinant proteins, various techniques known in the art can be used. For example, it may be possible to delete the lipidation signal peptide or replace it with a signal peptide that is not recognized by the cell producing the protein. U.S. Patent No. 10,300,122 B2 describes the use of this technique with fHBP.

[0214] The codon encoding the N-terminal cysteine can either be replaced with a codon encoding another amino acid or removed. For example, U.S. Pat. No. 10,300,122 B2 describes the insertion of an ATG (methionine) codon at the 5' end of the open reading frame encoding the mature fHBP protein. This results in a polypeptide lacking an N-terminal cysteine residue that can be lipidated. In addition, U.S. Pat. Nos. 9,724,402 B2 and 11,077,180 B2 disclose the production of non-lipidated fHBP in which the N-terminal cysteine residue is replaced with an amino acid other than cysteine.

[0215] The fHBP to be used in accordance with the present disclosure may be a naturally occurring protein or a non-naturally occurring protein. A "non-naturally occurring protein" refers to a "man-made protein" and, in contrast to a naturally occurring protein, encompasses an fHBP having heterologous components not found in nature. A non-naturally occurring protein may be a chimeric protein or a mutant protein. A "chimeric protein," in the context of the present disclosure, is intended to refer to a protein comprising two or more different components, each derived from a different fHBP (e.g., variant 1, 2, or 3). Mutations in mutant proteins may include amino acid substitutions, insertions, or deletions. In one embodiment, the mutation is an amino acid substitution.

[0216] A non-naturally occurring fHBP suitable for an immunogenic composition as disclosed herein is still capable of eliciting an immune response against the fHBP. In one embodiment, the non-naturally occurring fHBP to be used in accordance with the present disclosure may be a mutant fHBP. Introducing mutations, such as amino acid substitutions, can reduce or inhibit binding of the fHBP antigen to coagulation factor H (fH), which is normally present in an individual's blood. Reduced binding of mutant fHBP to fH can increase the amount of antigen available and accessible to the immune system. This can therefore improve the effectiveness and efficiency of the immune response against those antigens. Advantageously, mutant fHBPs can elicit anti-fHBP polyclonal antibodies directed against fHBP epitopes within the fH binding site, resulting in enhanced protective complement deposition activity compared to antibodies elicited by wild-type (WT) fHBP antigens targeting fHBP epitopes outside the fH binding site.

[0217] In some embodiments, the fHBP can be a mutant fHBP that includes at least one mutation that reduces or prevents binding of the fHBP to human factor H (fH).

[0218] Non-naturally occurring fHBPs contemplated for the immunogenic compositions as disclosed herein may exhibit decreased affinity for fH or increased thermal stability compared to the corresponding naturally occurring fHBP. Affinity for fH protein and thermal stability may be measured as disclosed in WO 2016 / 014719 A1 (as in Examples 1 or 3 herein).

[0219] For convenience and clarity, unless otherwise specifically indicated, the native or naturally occurring amino acid sequence of fHBP B24 (or fHBP ID1 or v.1 fHBP of N. meningitidis strain MC58) of sequence SEQ ID NO: 6 is chosen as the reference sequence for all naturally occurring and non-naturally occurring fHBP amino acid sequences herein. Accordingly, when referring to an amino acid residue position in fHBP, the position number used herein corresponds to the amino acid residue number of SEQ ID NO: 6 (fHBP B24). Consequently, position number 1 refers to the first amino acid residue shown in SEQ ID NO: 6, which is a cysteine. This remains true even if additional amino acids are added N-terminally to SEQ ID NO: 6 before this cysteine.

[0220] In one embodiment, mutations (e.g., amino acid substitutions) in fHBP A or B antigens used within the scope of this disclosure may be as disclosed in WO 2011 / 126863 A1, WO 2015 / 017817 A1, or WO 2016 / 014719 A1.

[0221] An immunogenic composition as disclosed herein can include a non-naturally occurring fHBP that differs in amino acid sequence from wild-type N. meningitidis fHBP by 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 to 15 amino acids, 15 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.

[0222] In some embodiments, the fHBP antigen may comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to a reference fHBP sequence.

[0223] Identity (e.g., percent homology) can be determined using various known sequence comparison tools, such as any homology comparison software that calculates pairwise sequence alignments, including, for example, the Blast software from the National Center of Biotechnology Information (NCBI), using default parameters. This identity is global identity, i.e., identity over the entire amino acid or nucleic acid sequence, rather than over a portion of it. Pairwise global alignment was defined by Needleman et al., Journal of Molecular Biology, 1970, pages 443-53, volume 48). For example, starting with a polypeptide sequence, one can compare it with another polypeptide sequence and use the EMBOSS-6.0.1 Needleman-Wunsch algorithm (available at http: / / emboss.sourceforge.net / apps / cvs / emboss / apps / needle.html) to find the optimal alignment of the two sequences along their entire length—a "global alignment."

[0224] The fHBP antigen to be used in the immunogenic compositions disclosed herein can be obtained as disclosed in WO 2016 / 014719 A1. The fHBP can be obtained as a recombinant protein from a recombinant expression vector (or construct) transfected into a production host cell, e.g., an E. coli strain. The composition of vectors suitable for the transfer and expression of fHBP-encoding nucleic acids can vary. Integration vectors can be conditionally replicating plasmids, suicide plasmids, bacteriophages, etc.

[0225] Constructs can include a variety of elements, including, for example, a promoter, a selectable genetic marker (e.g., a gene that confers resistance to an antibiotic, such as kanamycin, erythromycin, chloramphenicol, or gentamicin), an origin of replication site (to facilitate replication in a host cell, e.g., a bacterial host cell), etc. The choice of vector will depend on various factors, including the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and producing large amounts of a desired DNA sequence. Other vectors are suitable for expression in cells in culture. Selection of an appropriate vector is well within the skill of one in the art. Many such vectors are commercially available.

[0226] In one example, the vector may be an episomal plasmid-based expression vector containing a selectable drug resistance marker and elements that provide for autonomous replication in different host cells (e.g., in both E. coli and N. meningitidis). An example of such a "shuttle vector" is the plasmid pFPIO (Pagotto et al. (2000) Gene 244:13-19). The vector may provide for extrachromosomal maintenance in the host cell or may provide for integration into the host cell genome. Vectors are fully described in many publications well known to those skilled in the art, including, for example, Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons. The vector may provide for expression of a nucleic acid encoding a fHBP of interest, may provide for propagation of the nucleic acid of interest, or both.

[0227] Examples of vectors that can be used include, but are not limited to, those derived from recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA. For example, plasmid vectors such as pBR322, pUC 19 / 18, pUC 118, 119, and M13 mp series vectors can be used. pET21 is also an expression vector that can be used. Bacteriophage vectors include λgtl0, λgtll, λgtl8-23, λZAP / R, and EMBL series bacteriophage vectors. Additional vectors that can be used include, but are not limited to, pJB8, pCV103, pCV107, pCV108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16, and charomid 9 series vectors.

[0228] The recombinant expression vector can include a nucleotide sequence encoding fHBP operably linked to a transcriptional control element, such as a promoter. The promoter can be constitutive or inducible. The promoter can be adapted for use in prokaryotic or eukaryotic host cells.

[0229] The expression vector provides transcriptional and translational regulatory sequences, which can provide for inducible or constitutive expression, with the coding region operably linked under the transcriptional control of a transcriptional initiation region and a transcriptional and translational termination region. These regulatory regions can be native to the fHBP from which the fHBP of interest is derived, or can be derived from an exogenous source. Generally, the transcriptional and translational regulatory sequences can include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. The promoter can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7).

[0230] Expression vectors generally have convenient restriction sites located near the promoter sequence to allow for the insertion of a nucleic acid sequence encoding a protein of interest. Constructs (recombinant vectors) can be prepared by inserting a polynucleotide of interest into the construct backbone, typically by using DNA ligase-mediated attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Typically, homologous recombination can be achieved by attaching homologous regions to the vector adjacent to the desired nucleotide sequence, while site-specific recombination can be achieved by using sequences that promote site-specific recombination (e.g., Cre-lox, att sites, etc.). Nucleic acids containing such sequences can be added, for example, by oligonucleotide ligation or by polymerase chain reaction using primers containing both the homologous region and a portion of the desired nucleotide sequence.

[0231] In addition, the expression construct may contain additional elements. For example, the expression vector may have one or two replication systems, thus enabling it to be maintained in organisms, such as mammalian or insect cells for expression and prokaryotic hosts for cloning and amplification. In addition, the expression construct may contain a selectable marker gene that allows for the selection of transformed host cells. Selection genes are well known in the art and will vary depending on the host cell used.

[0232] 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.

[0233] Recombinant fHBP can be obtained in purified form from the culture by any purification method known in the art, for example, as described in this Examples section.

[0234] In one embodiment, fHBP A and / or fHBP B may be present in an immunogenic composition as disclosed herein in an amount of about 20 μg / dose to about 200 μg / dose, or about 25 μg / dose to about 180 μg / dose, or about 40 μg / dose to about 140 μg / dose, or about 50 μg / dose to about 120 μg / dose, or about 75 μg / dose to about 100 μg / dose. In one embodiment, fHBP A 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.

[0235] fHBP B The immunogenic composition as 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.

[0236] fHBP B can be a naturally occurring or non-naturally occurring fHBP. fHBP B can be a naturally occurring fHBP. In another embodiment, fHBP B can be a non-naturally occurring fHBP.

[0237] fHBP B can be a non-lipidated, non-naturally occurring fHBP.

[0238] The fHBP B protein can be a protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO: 3. A non-naturally occurring fHBP B01 protein is not 100% identical to fHBP B01 or SEQ ID NO: 3.

[0239] The non-naturally occurring fHBP B can 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 one exemplary embodiment, the fHBP B can be a mutant protein.

[0240] The non-naturally occurring fHBP B can be a mutant protein. The mutant fHBP B can be a non-lipidated protein.

[0241] The mutant fHBP B can differ in amino acid sequence from wild-type N. meningitidis fHBP B, such as fHBP B01, by 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 to 15 amino acids, 15 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.

[0242] Mutant fHBP B can be a mutant protein that includes at least one mutation that reduces or prevents binding of fHBP B to human factor H (fH).

[0243] The mutant fHBP B may comprise at least about 85% amino acid sequence identity with SEQ ID NO:3.

[0244] Mutant fHBP B can be a mutant protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 3. The mutant fHBP B protein is not 100% identical to fHBP B01 or SEQ ID NO: 3.

[0245] The mutant fHBP B may include 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.

[0246] Mutant fHBP B may comprise at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2011 / 051893 based on the numbering of the fHBP sequence identified as SEQ ID NO: 4 in WO 2011 / 051893 (fHBP B24 in the mature lipoprotein form): D37, K45, T56, E83, E95, E112, K122, V124, R127, T139, F141, D142, K143, 1198, S211, L213, K219, N43, D116, H119, S221, and K241.

[0247] Mutant fHBP B may contain at least one amino substitution at any one of the following positions as disclosed in WO2020 / 030782 based on the numbering of the fHBP sequence identified as SEQ ID NO: 2 in WO2020 / 030782 (fHBP B09 in the mature lipoprotein form): E211, S216, or E232.

[0248] Mutant fHBP B may include at least one of the following amino substitutions at any one of the following positions as disclosed in WO2020 / 030782 based on the numbering of the fHBP sequence identified as SEQ ID NO: 2 in WO2020 / 030782 (fHBP B09 in the mature lipoprotein form): E211A, S216R, or E232A.

[0249] Mutant fHBP B may contain at least one amino substitution at any one of the following positions as disclosed in WO2020 / 030782 based on the numbering of the fHBP sequence identified as SEQ ID NO: 6 in WO2020 / 030782 (fHBP B44 in the mature lipoprotein form): E214, S219, or E235.

[0250] Mutant fHBP B may include at least one of the following amino substitutions at any one of the following positions as disclosed in WO2020 / 030782 relative to the numbering of the fHBP sequence identified as SEQ ID NO: 2 in WO2020 / 030782 (fHBP B44 in the mature lipoprotein form): E214A, S219R, or E235A.

[0251] The mutant fHBP B protein may include at least one of the following amino acid substitutions at any one of the following positions as disclosed in WO2010046715 relative to the numbering of the fHBP sequence identified as SEQ ID NO: 1 in WO2010046715 (the 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, 287, 288, 289, 290, 291, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 4 8, 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 including amino acid numbers 103, 106, 107, 108, 180, 181, 183, 184, 185, 191, 193, 195, 262, 264, 266, 272, 274, 283, 286, 304 and 306 based on the numbering of the fHBP sequence identified as SEQ ID NO: 1 in WO2010046715.

[0252] The amino acid substitution for glutamine (Q38) at amino acid 38 can be a Q38R substitution (R: arginine). Other amino acids with positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, in some cases, a fHBP can include a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, or a Q38W substitution.

[0253] The amino acid substitution at amino acid 92 for glutamic acid (E92) can be an E92K substitution. Other amino acids with positively charged or aromatic side chains, such as arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, a fHBP variant can include an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution.

[0254] The amino acid substitution for arginine (R130) at amino acid 130 can be an R130G substitution (G:glycine). Other amino acids with negatively charged or aromatic side chains, such as aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, can also substitute for R130. Thus, for example, in some cases, a fHBP variant can include an R130D substitution, an R130E substitution, an R130F substitution, an R130Y substitution, or an R130W substitution.

[0255] The amino acid substitution for serine at amino acid 223 (S223) can be an S223R substitution (R: arginine). Other amino acids with positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, an fHbp variant contains an S223K, S223H, S223F, S223Y, or S223W substitution.

[0256] In one exemplary embodiment, the amino acid substitution for 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, such as isoleucine, valine, aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, can also be substituted for H248. Thus, for example, in some cases, a 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.

[0257] In another embodiment, mutant fHBP B may include at least the amino acid substitution H248L. Mutant fHBP B may include only the amino acid substitution H248L, based on the numbering of SEQ ID NO:6.

[0258] The mutant fHBP B may be a non-lipidated mutant fHBP B containing at least one 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.

[0259] Mutant fHBP B is a non-lipidated mutant fHBP comprising at least one amino acid substitution selected from the group consisting of a Q38R substitution, a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, a Q38W substitution, an E92K substitution, an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, an E92W substitution, an R130G substitution, an R130D substitution, an R130E substitution, an R130F substitution, an R130Y substitution, an R130W substitution, an S223R substitution, an S223K substitution, an S223H substitution, an S223F substitution, an S223Y substitution, an S223W substitution, an H248L substitution, an H248I substitution, an H248V substitution, an H248D substitution, an H248E substitution, an H248F substitution, an H248Y substitution, or an H248W substitution, based on the numbering of SEQ ID NO: 6. It could be B.

[0260] The mutant fHBP B can 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 can comprise only the amino acid substitution H248L, based on the numbering of SEQ ID NO: 6.

[0261] fHBP B can be a non-lipidated and mutant protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to SEQ ID NO: 3 and includes at least the amino acid substitution H248L based on the numbering of SEQ ID NO: 6. Non-lipidated mutant fHBP B can include only the amino acid substitution H248L based on the numbering of SEQ ID NO: 6.

[0262] The mutated 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.

[0263] In another embodiment, the mutated non-lipidated fHBP B may comprise or consist of SEQ ID NO:4.

[0264] The mutated 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.

[0265] In another embodiment, the mutated non-lipidated fHBP B may comprise or consist of SEQ ID NO:9.

[0266] fHBP B may have an isoelectric point (pI) above the PZC of the AlPO4 adjuvant.

[0267] fHBP B may have an isoelectric point (pI) in the range of about 5.0 to about 7.0, or 5.2 to about 6.5, or about 5.3 to about 60, or is about 5.5 or 5.46.

[0268] fHBP B may have an isoelectric point (pI) of about 5.46.

[0269] In some embodiments, the difference between the PZC of the AlPO4 adjuvant and the pI of fHBP B can 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.

[0270] The difference between the PZC of the AlPO4 adjuvant and the pI of fHBP B can be about 0.66 or about 1.16.

[0271] The difference between the PZC of the AlPO4 adjuvant and the pI of fHBP B may be about 0.66.

[0272] The difference between the PZC of the AlPO4 adjuvant and the pI of fHBP B may be about 0.96.

[0273] The difference between the PZC of the AlPO4 adjuvant and the pI of fHBP B may be about 1.16.

[0274] In one embodiment, fHBP B may be present in an immunogenic composition as disclosed herein in an amount of 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.

[0275] 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.

[0276] fHBP A The immunogenic compositions disclosed herein may comprise at least one fHBP A variant antigen. The at least one fHBP A may be a lipidated protein or a non-lipidated protein. The fHBP A may be a lipidated protein. The fHBP A may be a non-lipidated protein.

[0277] fHBP A and fHBP B can both be lipidated. In one embodiment, fHBP A and fHBP B can both be non-lipidated. Alternatively, fHBP A can be lipidated and fHBP B can be non-lipidated. Further alternatively, fHBP A can be non-lipidated and fHBP B can be lipidated.

[0278] fHBP A can be a naturally occurring or non-naturally occurring fHBP. fHBP A can be a naturally occurring fHBP. In another embodiment, fHBP A can be a non-naturally occurring fHBP.

[0279] fHBP A and fHBP B can both be naturally occurring fHBPs. fHBP A and fHBP B can both be non-naturally occurring fHBPs. Alternatively, fHBP A can be a naturally occurring fHBP and fHBP B can be a non-naturally occurring fHBP. Still alternatively, fHBP A can be a non-naturally occurring fHBP and fHBP B can be a naturally occurring fHBP.

[0280] fHBP A can be a non-lipidated, non-naturally occurring fHBP.

[0281] Both fHBP A and fHBP B can be non-lipidated, non-naturally occurring fHBPs.

[0282] fHBP A can be a protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO: 1. A non-naturally occurring fHBP A05 is not 100% identical to fHBP A05 or SEQ ID NO: 1.

[0283] The non-naturally occurring fHBP A can 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 one exemplary embodiment, the fHBP A can be a mutant protein.

[0284] The non-naturally occurring fHBP A can be a mutant protein. The mutant fHBP A can be a non-lipidated protein.

[0285] The mutant fHBP A can differ in amino acid sequence from a wild-type N. meningitidis fHBP A protein (e.g., fHBP A05) by 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 to 15 amino acids, 15 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.

[0286] The mutant fHBP A may comprise at least about 85% amino acid sequence identity with SEQ ID NO:1.

[0287] Mutant fHBP A can be a mutant protein that includes at least one mutation that reduces or prevents binding of fHBP A to human factor H (fH).

[0288] The mutant fHBP A can 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 is not 100% identical to fHBP A05 or SEQ ID NO: 1.

[0289] The mutant fHBP A may include at least one amino acid substitution selected from at least one 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 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.

[0290] Mutant fHBP A may comprise at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2011 / 051893 relative to the numbering of the fHBP sequence identified in WO 2011 / 051893 as SEQ ID NO: 5 (fHBP A19 in the mature lipoprotein form): D37, K45, T56, E83, E95, E112, S122, I124, R127, T139, F141, N142, Q143, L197, D210, R212, K218, N43, N116, K119, T220 and / or 240. In one embodiment, the amino acid deletion or substitution is as disclosed in WO 2011 / 051893.

[0291] Mutant fHBP A may contain at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2016 / 008960 based on 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 replaced with a different amino acid. For example, Leu-126 may be replaced with any of the other 19 naturally occurring amino acids. When a substitution is made, the replacing amino acid may, in some embodiments, be a simple amino acid such as glycine or alanine. In other embodiments, the replacing amino acids are conservative substitutions (e.g., they are made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). In other embodiments, the substitutions are non-conservative. In one embodiment, the substitutions at the designated residues are as follows: S32V; L126R; and / or E243A.

[0292] Mutant fHBP A may contain at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2016 / 008960, based on 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 replaced with a different amino acid. For example, Leu-123 may be replaced with any of the other 19 naturally occurring amino acids. When a substitution is made, the replacing amino acid may, in some embodiments, be a simple amino acid such as glycine or alanine. In other cases, the replacing amino acid is a conservative substitution (e.g., it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). In other embodiments, the substitution is non-conservative. In one embodiment, the substitution at the specified residue can be as follows: S32V; L123R; and / or E240A.

[0293] Mutant fHBP A can include at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2015 / 128480 based on the numbering of the fHBP sequence (fHBP A19 or v2.16 or ID16) identified in WO 2015 / 128480 as SEQ ID NO: 5: S32, V33, L39, L41, F69, V100, 1113, F122, L123, V124, S125, G126, L127, G128, S151, H239, and / or E240. In one embodiment, the mutated residues can be S32, V100, L123, V124, S125, G126, L127, G128, H239, and / or E240. Mutations at these residues result in a protein that exhibits better stability compared to wild-type fHBP A. In one embodiment, the residues mutated can be S32, L123, V124, S125, G126, L127, and / or G128. In one embodiment, the residues mutated can be S32, L123, V124, S125, G126, L127, and / or G128. In another embodiment, residues S32 and / or L123 can be mutated, e.g., S32V and / or L123. When one or more of V100, S125, and / or G126 are mutated, mutations at residues other than these three can also be introduced.

[0294] Although the specified residue can be deleted, preferably it is substituted with a different amino acid. For example, Ser-32 can be substituted with any of the other 19 naturally occurring amino acids. When substitutions are made, the replacing amino acid can, in some embodiments, be a simple amino acid such as glycine or alanine. In other embodiments, the replacing amino acid is a conservative substitution (e.g., it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). In other embodiments, the substitution is non-conservative. In some embodiments, the substitution does not use alanine.

[0295] Substitutions at the specified 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.

[0296] Mutant fHBP A can include at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2015 / 128480 based on the numbering of the fHBP sequence identified in WO 2015 / 128480 as SEQ ID NO: 17 (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 residues mutated can be S32, V103, L126, V127, S128, G129, L130, G131, H242, and / or E243. In one embodiment, the residues mutated may be S32, L126, V127, S128, G129, L130, and / or G131. In another embodiment, residues S32, L126, V127, S128, G129, L130, and / or G131 may be mutated, such as residues S32 and / or L126, e.g., S32V and / or L126R.

[0297] Although the specified residue can be deleted, preferably it is substituted with a different amino acid. For example, Ser-32 can be substituted with any of the other 19 naturally occurring amino acids. When substitutions are made, the replacing amino acid can, in some embodiments, be a simple amino acid such as glycine or alanine. In other embodiments, the replacing amino acid is a conservative substitution (e.g., it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). In other embodiments, the substitution is non-conservative. In some embodiments, the substitution does not use alanine.

[0298] Substitutions at the specified residues may be as follows: S32V; I33C; L39C; L41C; F72C; V103T; T116S; F125C; L126R; V127I; S128G or S128T; G129D; L130I; G131A; S154C; H242R; E243H.

[0299] The amino acid substitution for asparagine (N115) at amino acid 115 can be an N115I substitution (I: isoleucine). Other amino acids with non-polar, positively charged, or aromatic side chains, such as valine, leucine, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, in some cases, a fHBP can include a N115V substitution, a N115L substitution, a N115K substitution, a N115R substitution, a N115H substitution, a N115F substitution, a N115Y substitution, or a N115W substitution.

[0300] The amino acid substitution for 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, such as leucine, isoleucine, valine, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, a 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.

[0301] The amino acid substitution for serine (S128) at amino acid 128 can be an S128T substitution (T: threonine). Other amino acids with polar, charged, or aromatic side chains, such as methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, a 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.

[0302] 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 an L130R substitution (R: arginine).

[0303] Mutant fHBP A can include an amino acid substitution of valine (V131) at amino acid 131. Other amino acids with charged or aromatic side chains, such as glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, fHBP can include a V131E, V131K, V131R, V131H, V131F, V131Y, or V131W substitution.

[0304] 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).

[0305] Mutant fHBP A can include an amino acid substitution of lysine at position 219 (K219). Other amino acids with polar, negatively charged, or aromatic side chains, such as glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, fHBP can include a K219Q, K219D, K219E, K219F, K219Y, or K219W substitution.

[0306] The amino acid substitution for glycine acid (G220) at amino acid 220 can be a G220S substitution (S: serine). Other amino acids with polar, charged, or aromatic side chains, such as asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, a mutant fHBP A can include a G220N substitution, a G220Q substitution, a G220D substitution, a G220E substitution, a G220K substitution, a G220R substitution, a G220H substitution, a G220F substitution, a G220Y substitution, or a G220W substitution.

[0307] In one exemplary embodiment, the amino acid substitution at amino acid 130 for leucine (L130) can be an L130R substitution (R: arginine).

[0308] In one exemplary embodiment, the amino acid substitution for glycine at amino acid 133 (G133) can be a G133D substitution (D: aspartic acid).

[0309] In one exemplary embodiment, the amino acid substitution of glycine at position 220 (G220) can be a G220S substitution (S: serine).

[0310] In one exemplary embodiment, the amino acid substitution for phenylalanine at position 129 (F129) can be an F129S substitution (S: serine).

[0311] The mutant fHBP A may comprise at least one amino acid substitution selected from the group consisting of G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6. In another embodiment, the 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 the three amino acid substitutions G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6.

[0312] The mutant fHBP A may be a non-lipidated mutant fHBP A comprising at least one amino acid substitution 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.

[0313] The mutant fHBP A includes, based on the numbering of SEQ ID NO: 6, an N115I substitution, an N115V substitution, an N115L substitution, an N115K substitution, an N115R substitution, an N115H substitution, an N115F substitution, an N115Y substitution, an N115W substitution, a D121G substitution, a D121L substitution, a D121I substitution, a D121V substitution, a D121K substitution, a D121R substitution, a D121H substitution, a D121F substitution, a D121Y substitution, a D121W substitution, an S128T substitution, an S128M substitution, an S128N substitution, an S128D substitution, an S128E substitution, an S128K substitution, an S128R substitution, an S128H substitution, an S128F substitution, an S128Y substitution, an S128W substitution, a G220R substitution, a G220H substitution, a G220F substitution, a G220Y substitution, a G220W substitution, a G220S substitution, a G220N substitution, a G220Q substitution, a G220D substitution, a G220E substitution, a G220K substitution, a G220R substitution, a G220H substitution, a G220F substitution, a G220Y substitution, or a G220W substitution.

[0314] The mutant fHBP A can be a non-lipidated mutant fHBP A comprising at least one amino acid substitution selected from the group consisting of G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6. In another embodiment, the mutated non-lipidated fHBP A can 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 can comprise only the three amino acid substitutions G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6.

[0315] In one exemplary embodiment, fHBP A can be a non-lipidated or mutant protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 1 and includes at least one amino acid substitution selected from the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6. A mutated non-lipidated fHBP A protein can include 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, a non-lipidated mutant fHBP A protein can include only the three amino acid substitutions G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6.

[0316] In one embodiment, the mutated 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.

[0317] In another embodiment, the mutated non-lipidated fHBP A protein may comprise or consist of SEQ ID NO:2.

[0318] In one embodiment, the mutated 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.

[0319] In another embodiment, the mutated non-lipidated fHBP A protein may comprise or consist of SEQ ID NO:8.

[0320] fHBP A may have an isoelectric point (pI) ranging 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 5.9 or 5.86.

[0321] fHBP A may have an isoelectric point (pI) of about 5.86.

[0322] In some embodiments, the difference between the PZC of the AlPO4 adjuvant and the pI of fHBP A can 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.

[0323] The difference between the PZC of the AlPO4 adjuvant and the pI of fHBP A can be about 1.06 or about 1.56.

[0324] The difference between the PZC of the AlPO4 adjuvant and the pI of fHBP A can be about 1.06.

[0325] The difference between the PZC of the AlPO4 adjuvant and the pI of fHBP A may be about 1.36.

[0326] The difference between the PZC of the AlPO4 adjuvant and the pI of fHBP A may be about 1.56.

[0327] The fHBP A antigen may be present in the immunogenic compositions as disclosed herein in an amount of 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.

[0328] The fHBP A protein may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

[0329] NadA The immunogenic compositions of the present disclosure may further comprise at least one Neisserial adhesin A (NadA) protein.

[0330] Neisserial adhesin A (NadA, formerly known as GNA1994) is a surface-exposed trimeric protein that forms oligomers anchored to the outer membrane via its C-terminal transmembrane domain. NadA is expressed with a signal peptide and contains three major domains: (1) a COOH-terminal anchoring domain (β structure), which is also required for self-translocation to the bacterial surface; (2) a likely coiled-coil domain with a leucine zipper, which may mediate dimerization and oligomerization; and (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 sequences of NadA proteins from many N. meningitidis strains have been published, and the protein's activity as a neisserial adhesin has 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 stationary phase.

[0331] The NadA protein was included in the published genome sequence of N. meningitidis serogroup B strain MC58 as gene NMB1994 (GenBank accession number GI:7227256).

[0332] NadA polypeptides for use according to the present disclosure may be wild-type polypeptides or may be modified by amino acid substitutions, insertions or deletions, provided that the polypeptide is capable of eliciting an immune response against NadA.

[0333] The NadA protein to be used may be, for example, an N- and / or C-terminal truncated NadA or a NadA protein containing amino acid deletions or insertions, as disclosed in references WO 01 / 64920; WO 01 / 64922; or WO 03 / 020756.

[0334] The recombinant NadA protein to be used in the immunogenic compositions as disclosed herein may be a NadA1 variant. As shown in this example, NadA1 was shown to induce a strong hSBA response.

[0335] NadA1 can be obtained from the NadA sequence of MenB strain MC58.

[0336] The NadA protein may comprise or consist of the sequence of SEQ ID NO:7.

[0337] The NadA protein may 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.

[0338] The NadA protein may, for example, 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 SEQ ID NO: 7. If N-terminal residues are deleted, such deletions should not eliminate the ability of NadA to adhere to human epithelial cells.

[0339] The NadA protein may lack an N-terminal signal peptide, for example, the NadA protein may lack the N-terminal 23 amino acids of SEQ ID NO:7.

[0340] The NadA protein may lack its C-terminal membrane anchoring peptide, for example, the NadA protein may lack the C-terminal 55 amino acids of, for example, SEQ ID NO: 7.

[0341] NadA may be used in monomeric or oligomeric form, for example its trimeric form.

[0342] For example, the NadA protein can lack its C-terminal membrane anchor (e.g., deletion of residues 308-362 of strain MC58 (SEQ ID NO: 7)). Expression of NadA lacking its membrane anchor domain in E. coli can result in secretion of the protein into the culture supernatant while simultaneously removing the 23 amino acid signal peptide (e.g., deletion of residues 2-24 of SEQ ID NO: 7, leaving a 284 amino acid protein—SEQ ID NO: 5).

[0343] 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.

[0344] The NadA protein to be used in the immunogenic compositions disclosed herein may be obtained according to any recombinant technique known in the art, for example, as previously disclosed. The NadA protein may be obtained as a recombinant protein from a recombinant expression vector (or construct) transfected into a production host cell, for example, an E. coli strain. Recombinant NadA may be obtained in purified form from the culture by any purification method known in the art, for example, as described in this Examples section.

[0345] In one embodiment, 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, NadA protein may be present at or about 50 μg / dose.

[0346] dOMV The immunogenic compositions of the present disclosure may further comprise at least one detergent-extracted outer membrane vesicle (dOMV).

[0347] The immunogenic compositions as disclosed herein comprise detergent-extracted outer membrane vesicles (dOMVs), also known as outer membrane protein complexes (OMPCs). Detergent-extracted outer membrane vesicles are typically referred to as dOMVs or OMVs when used as antigens, and as OMPCs when used as protein carriers.

[0348] OMPC was used as the carrier protein platform for the polyribosylribitol phosphate (PRP) conjugate vaccines PedvaxHIB (Haemophilus influenzae type b vaccine) (Einhorn et al., Lancet (London, England). 1986;2(8502):299-302; Moro et al., The Journal of pediatrics. 2015;166(4):992-7) and VAXELIS (diphtheria, tetanus, pertussis, poliomyelitis, hepatitis B, and H. influenzae type b vaccine) (Syed, Pediatric drugs. 2017;19(1):69-80).

[0349] dOMVs are large proteolipid vesicles containing integral outer membrane proteins and the remnants of lipooligosaccharide (LOS) found in bacterial outer membranes (Helting, Acta Pathol Microbiol Scand 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 up to 50% of the total protein.

[0350] The Neisseria meningitidis porin proteins (Por) are antigenic determinants for serogroup typing. Two classes of porins, PorA and PorB, and antigenically distinct variants within each class resulting from sequence variation in the variable regions (VRs) of the porin gene, which encode the surface-exposed loops, have been identified.

[0351] Suitable dOMVs for the immunogenic compositions disclosed herein can be obtained from various MenB strains. The 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 a porin protein, such as a PorA or PorB protein, e.g., a PorA protein.

[0352] dOMVs can be obtained from MenB strains that express the PorA protein.

[0353] The dOMV may be obtained from a MenB strain expressing a PorA VR2 subtype, which may be a PorA VR2 type P1.2, P1.4, P1.7, P1.10 or P1.13 protein.

[0354] dOMVs can be obtained from MenB strains expressing the PorA VR2 type P1.2 protein.

[0355] dOMVs can be obtained from MenB strains expressing the PorA VR2 subtype and PorB P2.2a. dOMVs can be obtained from MenB strains expressing PorA VR2 P1.2 and PorB P2.2a.

[0356] The dOMV may comprise a PorA VR2 subtype and a PorB P2.2a. The dOMV may comprise a PorA VR2 P1.2 and a PorB P2.2a.

[0357] The dOMV may contain PorA VR2 P1.2 and PorB P2.2a and immunotype LOS L3,7. PorA and PorB may represent approximately 50% of the proteins in the dOMV.

[0358] dOMVs can be obtained from a single MenB strain or from a variety of different MenB strains, where the MenB strains may express different types of pore proteins, such as PorA proteins of the same subtype or different PorA protein subtypes or PorA and PorB proteins.

[0359] Usable MenB strains from which dOMVs provide the desired porin protein can be identified, for example, from the PubMLST database (https: / / pubmlst.org / ). For example, suitable MenB strains can be obtained by selecting MenB strains from epidemic outbreaks within such a database and then selecting, within such a subset, a MenB strain that has a gene encoding the porin protein of interest, such as the PorA VR2 P1.2 protein. One can then determine whether one or more of the selected strains effectively express the porin protein of interest using techniques known in the art.

[0360] Examples of MenB strains suitable for obtaining dOMVs according to the present disclosure include: NG H36, BZ 232, DK 353, B6116 / 77, BZ 163, 0085 / 00, NG P20, 0046 / 02, M11 40123, M12 240069, N5 / 99, 99M or M07 240677.

[0361] In one exemplary embodiment, the MenB strain can be MenB strain 99M, which expresses the PorA VR2 P1.2 protein subtype.

[0362] In one embodiment, the dOMV may comprise Porin A (PorA) VR2 subtype P1.2.

[0363] 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).

[0364] PorA may be present in a relative amount ranging from about 3% to about 15%, or from about 5% to about 9% or 10%, of the total protein present in the dOMV, and PorB may be present in a relative amount ranging from about 30% to about 70%, or from about 35% to about 65%, or from about 38% to about 58%, of the total protein present in the dOMV.

[0365] In one embodiment, dOMVs may be obtained by a detergent extraction method that employs at least a deoxycholate treatment step.

[0366] Suitable methods for obtaining dOMV may be as disclosed in Helting et al. (Acta Pathol Microbiol Scand C. 1981 Apr;89(2):69-78) or in Example 2 of U.S. Pat. No. 4,695,624. For example, the bacterial culture may be centrifuged to obtain a pellet, which may then be extracted with a detergent, such as deoxycholate or sodium dodecyl sulfate (SDS), under heating, for example, at about 50°C to about 60°C or about 56°C for a time ranging from about 10 to about 20 minutes, or for about 15 minutes. The resulting material may then be centrifuged, and the pellet may be further suspended and purified according to any method known in the art.

[0367] The dOMV may be present in an amount ranging from about 5 μg / dose to about 400 μg / dose, or from about 10 μg / dose to about 300 μg / dose, or from about 25 μg / dose to about 250 μg / dose, or from about 35 μg / dose to about 225 μg / dose, or from about 50 μg / dose to about 200 μg / dose, or from about 75 μg / dose to about 180 μg / dose, or from about 100 μg / dose to about 150 μg / dose, or from about 110 μg / dose to about 125 μg / dose.

[0368] In one embodiment, the dOMVs may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0369] Further antigens The immunogenic compositions of the present disclosure may include at least one additional antigen.

[0370] The additional antigen may be a saccharide antigen from N. 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 to a carrier protein.

[0371] In one embodiment, the additional antigen may be a combination of conjugates of MenA, MenC, MenW-135 and MenY capsular polysaccharides to a carrier protein.

[0372] Carrier proteins for different capsular polysaccharides can be different or the same. Examples of carrier proteins include inactivated bacterial toxins such as diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, exotoxin A from Escherichia coli (E. coli) LT, E. coli (E. coli) ST, and Pseudomonas aeruginosa. Bacterial outer membrane proteins, such as porin, transferrin-binding protein, lung detachment, pneumococcal surface protein A (PspA), or pneumococcal adhesin protein (PsaA), can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or purified protein derivatives of tuberculin (PPD), can also be used as carrier proteins. They can be CRM197 protein, tetanus, or diphtheria toxoid. In one embodiment, they are tetanus toxoid.

[0373] The conjugate may be a population comprising molecules with molecular weights ranging from 700 kDa to 1400 kDa or from 800 kDa to 1300 kDa.

[0374] Within each dose, the amount of each saccharide antigen can be 1-50 μg, measured as the mass of the saccharide. For example, a total of 40 μg of saccharide can be administered per dose. For example, 10 μg of each polysaccharide and about 55 μg of a carrier protein, such as tetanus toxoid protein, can be administered.

[0375] The additional antigen may be a combination of MenA, MenC, MenW-135, and MenY capsular polysaccharides, each conjugated to a tetanus toxoid carrier protein, with the MenA polysaccharide being conjugated to the tetanus toxoid carrier via an adipic acid dihydrazide (ADH) linker, while the MenC, MenW-135, and MenY polysaccharides are each directly conjugated to the tetanus toxoid carrier (TT).

[0376] In one embodiment, the additional antigen may be a combination of conjugates of MenA, MenC, MenW-135 and MenY capsular polysaccharides to tetanus toxoid carrier protein. In an exemplary embodiment, the conjugated saccharide antigens from N. meningitidis serogroups A, C, W135 and / or Y may be as disclosed in WO 2018 / 045286 A1 or WO 2002 / 058737 A2.

[0377] In one embodiment, the additional antigen is that of the commercially available MenACYW-TT conjugate vaccine MENQUADFI®.

[0378] immunogenic composition The immunogenic compositions disclosed herein may include a combination of meningococcal antigens, including at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

[0379] The immunogenic compositions disclosed herein can include a combination of Neisseria meningitidis serogroup B antigens, including at least one factor H binding protein (fHBP) A, at least one fHBP B, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV). fHBP A and / or fHBP B can be non-lipidated.

[0380] fHBP A can be a mutant protein comprising at least about 85% identity to SEQ ID NO:1, and / or fHBP B can be a mutant protein comprising at least about 85% identity to SEQ ID NO:3.

[0381] fHBP A can be a mutant protein comprising at least one mutation that reduces or prevents binding of fHBP A to human factor H (fH), and / or fHBP B can be a mutant protein comprising at least one mutation that reduces or prevents binding of fHBP B to human factor H (fH).

[0382] fHBP A may comprise at least one amino acid substitution selected from at least one 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 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 comprises or consists of SEQ ID NO: 2, or comprises or consists of SEQ ID NO: 8, and / or fHBP The B protein 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 comprises or consists of SEQ ID NO: 4, or comprises or consists of SEQ ID NO: 9.

[0383] 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 at about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

[0384] 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.

[0385] 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 at about 50 μg / dose.

[0386] The dOMV may comprise Porin A (PorA).

[0387] The dOMV may be present in an amount ranging from about 5 μg / dose to about 400 μg / dose, or from about 10 μg / dose to about 300 μg / dose, or from about 25 μg / dose to about 250 μg / dose, or from about 35 μg / dose to about 225 μg / dose, or from about 50 μg / dose to about 200 μg / dose, or from about 75 μg / dose to about 180 μg / dose, or from about 100 μg / dose to about 150 μg / dose, or from about 110 μg / dose to about 125 μg / dose, or at about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0388] The composition may include an adjuvant, such as an aluminum-based adjuvant selected from the group including aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum sulfate adjuvant, aluminum hydroxyphosphate adjuvant, potassium aluminum sulfate adjuvant, aluminum hydroxycarbonate, a combination of aluminum hydroxide and magnesium hydroxide, and mixtures thereof, such as aluminum phosphate adjuvant.

[0389] The composition may have a pH that is 0.6 to 2.9 units from the PZC of the AlPO4 adjuvant, or 1.2 to 2.9 units from the PZC of the adjuvant.The composition may have a pH that is 0.6 to 2.9 units from the PZC of the AlPO4 adjuvant, or 1.0 to 2.8, or 1.2 to 2.5, or 1.4 to 2.1 units from the PZC of the adjuvant.

[0390] The composition may have a pH that is 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 from the PZC of the AlPO4 adjuvant.

[0391] In some embodiments, the AlPO4 adjuvants of the present disclosure are used in compositions having a pH such that the difference between the PZC of the AlPO4 adjuvant and the pH of the composition ranges from about 1.0 to about 2.9, or from about 1.2 to about 2.9.

[0392] The composition may have a pH that is at least 1.2 units from the PZC of the AlPO4 adjuvant.

[0393] The composition may have a pH that is 2.9 units or less from the PZC of the AlPO4 adjuvant.

[0394] The composition may have a pH that is 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 from the PZC of the AlPO4 adjuvant.

[0395] The composition may have a pH that is 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 from the PZC of the AlPO4 adjuvant.

[0396] The composition may have a pH that is 1.2, 1.5, 1.7, 2.2, 2.5 or 2.7 units from the PZC of the AlPO4 adjuvant.

[0397] The composition may have a pH that is 1.2, 1.3, 1.4, 1.5, 1.6 or 1.7 units from the PZC of the AlPO4 adjuvant.

[0398] The composition may have a pH that is 1.2, 1.5, or 1.7 units from the PZC of the AlPO4 adjuvant.

[0399] The composition may have a pH of about 5.5 to about 7.0. The composition may have a pH of 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.

[0400] The composition may have a pH of about 5.5, 6.0, 6.5 or about 7.0.

[0401] The composition may have a pH of about 6.0.

[0402] An AlPO4 adjuvant may have a PZC of about 4.5.

[0403] The composition may have a pH within 1.5 units of the PZC of the AlPO4 adjuvant.

[0404] The composition may comprise or consist of about 25 to about 100 μg / dose of non-lipidated fHBP A protein comprising or consisting of SEQ ID NO: 2, about 25 to about 100 μg / dose of non-lipidated fHBP B protein comprising or consisting of SEQ ID NO: 4, about 25 to about 100 μg / dose of NadA protein comprising or consisting of SEQ ID NO: 5, about 20 to about 150 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 600 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer, pH 6.0.

[0405] The composition may comprise or consist of about 25 to about 100 μg / dose of non-lipidated fHBP A protein comprising or consisting of SEQ ID NO: 8, about 25 to about 100 μg / dose of non-lipidated fHBP B protein comprising or consisting of SEQ ID NO: 9, about 25 to about 100 μg / dose of NadA protein comprising or consisting of SEQ ID NO: 5, about 20 to about 150 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 600 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer, pH 6.0.

[0406] The composition may further comprise at least a conjugated capsular saccharide from one or more of Neisseria meningitidis serogroups A, C, W135 and / or Y.

[0407] Also disclosed are vaccines comprising the compositions as described herein.

[0408] A composition or vaccine as disclosed herein may be for use in protecting against meningococcal infection or may be for use in inducing an immune response against meningococcal bacteria.

[0409] Further disclosed are compositions comprising or consisting of mRNA encoding a fHBP A protein having at least about 85%, at least about 90%, at least 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:2; mRNA encoding a fHBP B protein having 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; mRNA encoding a NadA protein having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:5; and a dOMV from MenB expressing PorA VR2 P1.2.

[0410] Further disclosed are compositions comprising or consisting of mRNA encoding a fHBP A protein having at least about 85%, at least about 90%, at least 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:8; mRNA encoding a fHBP B protein having 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; mRNA encoding a NadA protein having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:5; and a dOMV from MenB expressing PorA VR2 P1.2.

[0411] formulation The immunogenic compositions as disclosed herein can be formulated into preparations in solid, semi-solid, or liquid form, such as tablets, capsules, powders, aerosols, solutions, suspensions, or emulsions. Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The compositions are formulated based on the mode of delivery, including, for example, that the compositions can be formulated for delivery by parenteral delivery, such as intramuscular, intradermal, or subcutaneous injection.

[0412] The immunogenic compositions may be administered by any suitable route, such as mucosally (e.g., intranasally or sublingually), parenterally (e.g., intramuscular, subcutaneous, transdermal, or intradermal routes), or orally. Typical routes of administration of such compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, and intranasal. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques. In some embodiments, the compositions may be administered by transdermal, subcutaneous, intradermal, or intramuscular routes.

[0413] In one embodiment, the immunogenic composition as disclosed herein may be formulated to be administered by intramuscular, intradermal, or subcutaneous routes. In one embodiment, the immunogenic composition may be formulated to be administered by intramuscular route.

[0414] The immunogenic composition may be formulated with any pharmaceutically acceptable excipient. The composition may include at least one inert diluent or carrier. One 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 edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. The immunogenic composition as described herein may optionally include pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, non-essential amino acids, L-arginine hydrochloride, sucrose, D-trehalose dehydrate, sorbitol, tris(hydroxymethyl)aminomethane, and / or urea. In addition, the vaccine composition may optionally contain pharmaceutically acceptable excipients, including, for example, diluents, binders, stabilizers and preservatives.

[0415] The composition may be in the form of a liquid intended for delivery by injection, such as a solution, emulsion, or suspension. Compositions intended for administration by injection may contain at least one of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents. The liquid composition as disclosed herein may include at least one of the following: sterile diluents such as water for injection, saline solution, for example, physiological saline, Ringer's solution, isotonic saline, fixed oils such as synthetic monoglycerides or diglycerides, polyethylene glycols, glycerin, propylene glycol, or other solvents that can serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose; and cryoprotectants such as sucrose or trehalose.

[0416] In another embodiment, the compositions of the present disclosure may have a pH ranging from about 5.5 to about 7.0.

[0417] 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 a composition as disclosed herein can be about 6.0. A stable pH can be maintained by the use of a buffer.

[0418] In one embodiment, the composition of the present disclosure may further comprise a buffer, such as Tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer, or histidine buffer.

[0419] The composition may include a sodium acetate buffer.

[0420] The sodium acetate buffer may be present at a concentration in the range of about 10 mM to about 300 mM, or about 10 mM to about 250 mM, or about 20 mM to about 250 mM, or about 20 mM to about 150 mM, or about 20 mM to about 130 mM, or about 30 mM to about 120 mM, or about 40 mM to about 100 mM, or about 50 mM to about 80 mM, or about 50 mM to about 60 mM, or at a concentration of, for example, about 50 mM.

[0421] The immunogenic composition can be isotonic with respect to a mammal, such as a human.

[0422] The immunogenic composition may also contain one or several additional salts, such as sodium salts, calcium salts, or magnesium salts. The sodium salt may be selected from the group including sodium chloride and sodium phosphate. The sodium salt may be sodium chloride. The calcium salt may be calcium chloride salt. The magnesium salt may be magnesium chloride salt.

[0423] The sodium salt may be present at a concentration ranging from about 10 mM to about 300 mM, or from about 30 mM to about 280 mM, or from about 50 mM to about 250 mM, or from about 60 mM to about 220 mM, or from about 80 mM to about 200 mM, or from about 100 mM to about 180 mM, or from about 120 mM to about 160 mM, or may be at a concentration of, for example, about 150 mM.

[0424] Calcium or magnesium can be present in an amount ranging from about 1 mM to about 15 mM or from about 5 mM to about 10 mM.

[0425] Immunogenic compositions for parenteral administration can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Compositions for injection are, for example, sterile.

[0426] The immunogenic composition can be sterilized by conventional sterilization techniques, such as ultraviolet or gamma irradiation, or can be sterile filtered. The composition obtained after sterile filtration can be packaged and stored in liquid or lyophilized form. Lyophilized compositions can be reconstituted with a sterile aqueous carrier before administration. Dry compositions can include stabilizers such as mannitol, sucrose, or dodecyl maltoside, as well as mixtures thereof, such as lactose / sucrose mixtures, sucrose / mannitol mixtures, etc.

[0427] The compositions as disclosed herein are administered to an individual in need thereof in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent used; the metabolic stability and 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; the drug combination; the severity of the particular disorder or condition; and the subject under therapy.

[0428] The immunogenic compositions of the present disclosure may comprise or consist of:

[0429] - a non-lipidated mutant fHBP A comprising or consisting of SEQ ID NO: 2, a non-lipidated mutant fHBP B comprising or consisting of SEQ ID NO: 4, a NadA protein comprising or consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, and an AlPO4 adjuvant selected as having a PZC of about 4.3, which composition may comprise 50 mM acetate buffer and pH 6.0, or

[0430] - 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, a dOMV from MenB expressing PorA VR2 P1.2, an AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0431] - about 25 to about 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 to about 100 μg / dose of 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 an AlPO4 adjuvant selected as having a PZC of about 4.3, in 50 mM acetate buffer and at pH 6.0; or

[0432] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0433] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant with a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0434] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0435] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0436] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0437] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 200 μg / dose of an AlPO4 adjuvant selected as having a PZC of about 4.3, in 50 mM acetate buffer and at pH 6.0; or

[0438] - about 75 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 75 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 75 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 75 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 300 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer, pH 6.0, or

[0439] - 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 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0440] - 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO adjuvant with a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0441] - 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 a MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer, pH 6.0.

[0442] In one embodiment, the immunogenic composition may comprise or consist of about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer, pH 6.0.

[0443] The immunogenic compositions of the present disclosure may comprise or consist of:

[0444] - a non-lipidated mutant fHBP A comprising or consisting of SEQ ID NO: 2, a non-lipidated mutant fHBP B comprising or consisting of SEQ ID NO: 4, a NadA protein comprising or consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, and an AlPO4 adjuvant selected as having a PZC of about 4.5, which composition may comprise 50 mM acetate buffer and pH 6.0, or

[0445] - 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, a dOMV from MenB expressing PorA VR2 P1.2, an AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0446] - about 25 to about 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 to about 100 μg / dose of 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 an AlPO4 adjuvant selected as having a PZC of about 4.5, in 50 mM acetate buffer and at pH 6.0; or

[0447] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0448] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO adjuvant with a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0449] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0450] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0451] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0452] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 200 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0453] - about 75 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 75 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 75 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 75 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 300 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0454] - 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 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0455] - 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant with a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0456] - 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 a MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer, pH 6.0.

[0457] In one embodiment, the immunogenic composition may comprise or consist of about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer, pH 6.0.

[0458] The immunogenic compositions of the present disclosure may comprise or consist of:

[0459] - a non-lipidated mutant fHBP A comprising or consisting of SEQ ID NO: 2, a non-lipidated mutant fHBP B comprising or consisting of SEQ ID NO: 4, a NadA protein comprising or consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, and an AlPO4 adjuvant selected as having a PZC of about 4.8, which composition may comprise 50 mM acetate buffer and pH 6.0, or

[0460] - 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, a dOMV from MenB expressing PorA VR2 P1.2, an AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0461] - about 25 to about 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 to about 100 μg / dose of 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 an AlPO4 adjuvant selected as having a PZC of about 4.8, in 50 mM acetate buffer and at pH 6.0; or

[0462] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0, or

[0463] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant with a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0464] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0465] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0466] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0467] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 200 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0468] - about 75 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 75 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 75 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 75 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 300 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0469] - 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 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0, or

[0470] - 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant with a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0471] - 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 a MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0.

[0472] In one embodiment, the immunogenic composition may comprise or consist of about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0.

[0473] The immunogenic compositions of the present disclosure may comprise or consist of:

[0474] - a non-lipidated mutant fHBP A comprising or consisting of SEQ ID NO: 8, a non-lipidated mutant fHBP B comprising or consisting of SEQ ID NO: 9, a NadA protein comprising or consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, and an AlPO4 adjuvant selected as having a PZC of about 4.3, which composition may comprise 50 mM acetate buffer and pH 6.0, or

[0475] - a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, a NadA protein consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, an AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0476] - about 25 to about 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 to about 100 μg / dose of 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 as having a PZC of about 4.3, in 50 mM acetate buffer and at pH 6.0; or

[0477] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0478] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant with a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0479] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0480] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0481] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer, pH 6.0, or

[0482] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 200 μg / dose of an AlPO4 adjuvant selected as having a PZC of about 4.3, in 50 mM acetate buffer and at pH 6.0; or

[0483] - about 75 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 75 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 75 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 75 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 300 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0484] - 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 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of an AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0485] - 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO adjuvant with a PZC of about 4.3, 50 mM acetate buffer and pH 6.0, or

[0486] - 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 a MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer, pH 6.0.

[0487] In one embodiment, the immunogenic composition may comprise or consist of about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.3, 50 mM acetate buffer, pH 6.0.

[0488] The immunogenic compositions of the present disclosure may comprise or consist of:

[0489] - a non-lipidated mutant fHBP A comprising or consisting of SEQ ID NO: 8, a non-lipidated mutant fHBP B comprising or consisting of SEQ ID NO: 9, a NadA protein comprising or consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, and an AlPO4 adjuvant selected as having a PZC of about 4.5, which composition may comprise 50 mM acetate buffer and pH 6.0, or

[0490] - a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, a NadA protein consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, an AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0491] - about 25 to about 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 to about 100 μg / dose of 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 as having a PZC of about 4.5, in 50 mM acetate buffer and at pH 6.0; or

[0492] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0493] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant at a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0494] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0495] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of an AlPO4 adjuvant selected as having a PZC of about 4.5, in 50 mM acetate buffer and at pH 6.0; or

[0496] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of an AlPO4 adjuvant selected as having a PZC of about 4.5, in 50 mM acetate buffer and at pH 6.0; or

[0497] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 200 μg / dose of an AlPO4 adjuvant selected as having a PZC of about 4.5, in 50 mM acetate buffer and at pH 6.0; or

[0498] - about 75 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 75 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 75 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 75 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 300 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0499] - 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 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of an AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0500] - 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO adjuvant at a PZC of about 4.5, 50 mM acetate buffer and pH 6.0, or

[0501] - 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 a MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer, pH 6.0.

[0502] In one embodiment, the immunogenic composition may comprise or consist of about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.5, 50 mM acetate buffer, pH 6.0.

[0503] The immunogenic compositions of the present disclosure may comprise or consist of:

[0504] - a non-lipidated mutant fHBP A comprising or consisting of SEQ ID NO: 8, a non-lipidated mutant fHBP B comprising or consisting of SEQ ID NO: 9, a NadA protein comprising or consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, and an AlPO4 adjuvant selected as having a PZC of about 4.8, which composition may comprise 50 mM acetate buffer and pH 6.0, or

[0505] - a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, a NadA protein consisting of SEQ ID NO: 5, a dOMV from MenB expressing PorA VR2 P1.2, an AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0506] - about 25 to about 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 to about 100 μg / dose of 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 as having a PZC of about 4.8, in 50 mM acetate buffer and at pH 6.0; or

[0507] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0, or

[0508] - about 25 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant with a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0509] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of an AlPO4 adjuvant selected as having a PZC of about 4.8, in 50 mM acetate buffer and at pH 6.0; or

[0510] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0511] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0512] - about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 a MenB strain expressing PorA VR2 P1.2, about 200 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0, or

[0513] - about 75 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 75 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 75 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 75 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 300 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0, or

[0514] - 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 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0, or

[0515] - 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 a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant with a PZC of about 4.8, 50 mM acetate buffer and pH 6.0, or

[0516] - 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 a MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0.

[0517] In one embodiment, the immunogenic composition may comprise or consist of about 50 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 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 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of AlPO4 adjuvant selected as having a PZC of about 4.8, 50 mM acetate buffer, pH 6.0.

[0518] The dose may range from about 0.1 mL to about 1 mL, for example, from about 0.2 mL to about 0.8 mL, from about 0.4 mL to about 0.6 mL, or may be about 0.5 mL.

[0519] In one embodiment, the present disclosure relates to a container comprising a composition as disclosed herein. The container may contain an immunogenic composition comprising a combination of meningococcal antigens, the combination comprising at least one factor H binding protein (fHBP) A, at least one fHBP B, and an aluminum hydroxyphosphate (AlPO4) adjuvant, where the AlPO4 adjuvant is selected as having a point of zero charge (PZC) below 5.

[0520] The container may further comprise at least one Neisserial adhesin A (NadA) protein and / or at least one detergent-extracted outer membrane vesicle (dOMV).

[0521] The container may contain a composition of the present disclosure as detailed above.

[0522] The container may contain a composition comprising or consisting of about 25-100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25-100 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25-100 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 20-250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100-800 μg / dose of an AlPO adjuvant selected as having a point of zero charge (PZC) below 5, 50 mM acetate buffer, pH 6.0.

[0523] The container may contain a composition comprising or consisting of about 25-100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25-100 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25-100 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 20-250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100-800 μg / dose of an AlPO adjuvant selected as having a point of zero charge (PZC) below 5, 50 mM acetate buffer, pH 6.0.

[0524] In addition, the container may contain further antigens as detailed above. Alternatively, the further antigens may be packaged in a separate container.

[0525] The container may be a vial. The vial may be a multi-dose vial or a single-dose vial. Suitable vials may be small glass or plastic containers that are closed with the most suitable stopper and seal.

[0526] Alternatively, the container can be a pre-filled syringe. The pre-filled syringe can include a syringe barrel that stores the liquid composition as disclosed herein. A gasket and a plunger are inserted into the syringe barrel. The gasket seals the syringe barrel to prevent leakage of the liquid drug, and the plunger slides along the gasket. Various pre-filled syringes are known in the art, for example, as described in U.S. Patent No. 10,625,025 or WO 2013 / 046855.

[0527] When the composition of the present disclosure is to be mixed with another vaccine composition, such as a tetravalent MenACWY conjugate composition, for injection, both compositions can be packaged in a single container, such as a single vial or prefilled syringe, or a dual-chamber syringe. Dual-chamber syringes, also known as sequential or bypass syringes, can include a single syringe barrel separated into two compartments, a proximal and a distal compartment, by a septum. Depressing the syringe plunger forces the two vaccine compositions in the distal compartment to mix. Various dual-chamber syringes are known in the art, for example, as described in U.S. Pat. No. 10,695,505. Dual-chamber syringes can also be used when the vaccine composition is formulated in a dry form, such as a lyophilized form, and stored with a liquid medium for reconstitution. In this case, one chamber stores the dried vaccine, and the second chamber stores the liquid for reconstitution and injection.

[0528] In some embodiments, the present disclosure relates to a vaccine comprising an immunogenic composition as disclosed herein.

[0529] The immunogenic composition of the present disclosure can be a vaccine.

[0530] Kit of Parts A kit-of-parts is also disclosed.

[0531] The kit of parts may include at least two containers, a first container containing at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and a second container containing an aluminum hydroxyphosphate (AlPO4) adjuvant selected as having a point of zero charge (PZC) below 5.

[0532] The kit of parts may include at least a further container containing at least one detergent-extracted outer membrane vesicle (dOMV) and / or at least one Neisserial adhesin A (NadA) protein. The dOMV and NadA protein may be provided in separate containers.

[0533] Alternatively, the kit of parts may include at least a first container containing at least one factor H binding protein (fHBP) A, at least one factor H binding protein (fHBP) B, and an aluminum hydroxyphosphate (AlPO4) adjuvant selected as having a point of zero charge (PZC) below 5, and a second container containing 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.

[0534] Alternatively, the kit of parts may comprise at least a first container containing at least one factor H binding protein (fHBP) A, at least one factor H binding protein (fHBP) B, an aluminum hydroxyphosphate (AlPO4) adjuvant selected as having a point of zero charge (PZC) below 5, at least one detergent-extracted outer membrane vesicle (dOMV) and at least one Neisseria adhesin A (NadA) protein, and a second container containing a further antigen.

[0535] Alternatively, each of the antigens, fHBP A, fHBP B, NadA and dOMV, and the AlPO4 adjuvant selected as having a point of zero charge (PZC) below 5, can be stored in separate containers. Further alternatively, the antigens and AlPO4 adjuvants can be combined in different combinations. Any kind of combinations can be envisaged: 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, etc.

[0536] fHBP A and B, AlPO4 adjuvant, NadA protein and dOMV are as detailed above.

[0537] In one embodiment, the additional antigen may be a combination of conjugated MenACWY polysaccharides.

[0538] The conjugated MenACWY polysaccharide may be as detailed above.

[0539] In one embodiment, the kit of parts may comprise a first container comprising an immunogenic composition as disclosed herein and a second container comprising a combination of conjugated MenACWY polysaccharides.

[0540] The antigen and AlPO4 of the immunogenic compositions of the present disclosure may be prepared and stored in separate containers or vials, which may then be mixed at the time of administration to an individual.

[0541] Antigens can be stored in liquid formulations or in dry form. When formulated in dry form, an additional container containing an injection solution can be added, and this injection solution can be used to resuspend and mix different antigens. Suitable injection solution carriers can include buffers. The injection solution can include an AlPO4 adjuvant.

[0542] In one embodiment, the kit-of-parts container may contain the antigen, which may be in a dried form. The antigen may be lyophilized in a cake or as a micropellet.

[0543] The kit may optionally include a container containing a physiologically injectable medium, which may be used to resuspend or dissolve the antigen in a dry form.

[0544] Manufacturing method The present disclosure provides a method for producing 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 one factor H binding protein (fHBP) B, and an AlPO4 adjuvant, the method comprising at least: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AIPO4 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 combinations being carried out in any order; The present invention relates to a method comprising:

[0545] In this way, immunogenic compositions can be obtained.

[0546] Combining the AlPO4 adjuvant with fHBP A and fHBP B can be performed in any order. For example, the AlPO4 adjuvant can be combined with fHBP A, then fHBP B can be added, or the AlPO4 adjuvant can be combined with fHBP B, then fHBP A can be added, or fHBP A and fHBP B can be combined, then AlPO4 can be added, or the AlPO4 adjuvant can be combined with both fHBP A and fHBP B at the same time.

[0547] In step b), fHBP A and fHBP B may be first combined, and then the AlPO4 adjuvant may be added.

[0548] Alternatively, in step b), the AlPO4 adjuvant and fHBP A may be combined first, and then fHBP B may be added.

[0549] Alternatively, in step b), the AlPO4 adjuvant and fHBP B may be combined first, and then fHBP A may be added.

[0550] Alternatively, in step b), the first part of the AlPO4 adjuvant and fHBP B may be combined in a first mixture, the second part of the AlPO4 adjuvant and fHBP A may be combined in a second mixture, and then the first and second mixtures may be combined.

[0551] Alternatively, in step b), the AlPO4 adjuvant can be combined with both fHBP A and fHBP B at the same time.

[0552] The method of the present disclosure may further comprise the step of adding at least one antigen selected from NadA protein and dOMV. The combining may be performed in any order.

[0553] For example, NadA protein and / or dOMV may be added before or after combining AlPO with fHBP A and fHBP B. NadA and dOMV may each be added in separate steps, or may be combined and then added in a single step.

[0554] In some embodiments, in step b), fHBP A, fHBP B, NadA protein and dOMV may be combined in any order, followed by the addition of AlPO4 adjuvant.

[0555] In some embodiments, in step b), the AlPO4 adjuvant may be fractionated into multiple fractions (2, 3 or 4) and one fraction may be added for each antigen: fHBP A, fHBP B, NadA protein and dOMV, and then the antigens with AlPO4 may be combined together in any order.

[0556] Alternatively, step b) may involve combining AlPO4 adjuvant with a combination of fHBP A, fHBP B and NadA proteins. The dOMVs may be added in a subsequent step.

[0557] Alternatively, step b) may involve combining AlPO4 adjuvant with a combination of fHBP A, fHBP B and dOMV. NadA protein may be added in a subsequent step.

[0558] The method for producing the immunogenic composition of the present disclosure comprises at least: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AIPO4 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, in any order. may include:

[0559] The Neisseria meningitidis antigens fHBP A, fHBP B, NadA protein, dOMV may be filtered, for example sterile filtered, for example through a 0.22 μm filter, before being combined with the AlPO 4 adjuvant.

[0560] Following combining the AlPO4 adjuvant, fHBP A, fHBP B, NadA protein and dOMV, the resulting combination may then be dispensed into syringes or vials.

[0561] The present disclosure provides a method for preparing an immunogenic composition comprising a N. meningitidis fHBP B antigen, the method inducing an enhanced immune response against a N. meningitidis serogroup B strain expressing an fHBP B heterologous to the fHBP B antigen of the composition, the method comprising at least: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AlPO4 adjuvant selected in step a) with said fHBP B; c) obtaining said immunogenic composition; The present invention relates to a method comprising:

[0562] This method makes it possible to obtain an immunogenic composition capable of inducing an enhanced immune response against N. meningitidis serogroup B strains expressing fHBP B heterologous to the fHBP B of the composition.

[0563] According to another of its objects, the present disclosure provides a method for preparing an immunogenic composition comprising a N. meningitidis fHBP B antigen, the method inducing an enhanced immune response against a N. meningitidis serogroup B strain expressing an fHBP B homologous to said fHBP B antigen of said composition, the method comprising at least: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AlPO4 adjuvant selected in step a) with said fHBP B; c) obtaining said immunogenic composition; The present invention relates to a method comprising:

[0564] This method makes it possible to obtain an immunogenic composition capable of inducing an enhanced immune response against N. meningitidis serogroup B strains that express fHBP B homologous to the fHBP B of the composition.

[0565] The composition may further comprise at least one of fHBP A, NadA protein, or dOMV.

[0566] According to another of its objects, the present disclosure provides a method for preparing an immunogenic composition comprising a N. meningitidis fHBP A antigen, the method inducing an enhanced immune response against a N. meningitidis serogroup B strain expressing a fHBP A heterologous to the fHBP A antigen of the composition, the method comprising at least: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AlPO4 adjuvant selected in step a) with said fHBP A; c) obtaining said immunogenic composition; The present invention relates to a method comprising:

[0567] This method makes it possible to obtain an immunogenic composition capable of inducing an enhanced immune response against N. meningitidis serogroup B strains expressing a fHBP A heterologous to the fHBP A of the composition.

[0568] According to another of its objects, the present disclosure provides a method for preparing an immunogenic composition comprising a N. meningitidis fHBP A antigen, the method inducing an enhanced immune response against a N. meningitidis serogroup B strain expressing an fHBP A homologous to said fHBP A antigen of said composition, the method comprising at least a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AlPO4 adjuvant selected in step a) with said fHBP A; c) obtaining said immunogenic composition; The present invention relates to a method comprising:

[0569] This method makes it possible to obtain an immunogenic composition capable of inducing an enhanced immune response against N. meningitidis serogroup B strains that express a fHBP A homologous to the fHBP A of the composition.

[0570] The composition may further comprise at least one of fHBP B, NadA protein, or dOMV.

[0571] The method of the present disclosure may further include adding at least one antigen selected from fHBP A, NadA protein, and dOMV. Adding the AlPO4 adjuvant, fHBP B, fHBP A, NadA protein, and dOMV may be performed in any order.

[0572] The method of producing the immunogenic composition of the present disclosure includes: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AIPO4 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 combinations being carried out in any order. It may include at least:

[0573] The fHBP A and B, NadA protein and dOMV may be as detailed above.

[0574] The fHBP A and B, NadA protein and dOMV may be sterile filtered before combining together and with the AlPO4 adjuvant.

[0575] The methods of the present disclosure may also include the step of adding an additional antigen, which may be a combination of MenACWY polysaccharide conjugated to a protein carrier as detailed above.

[0576] The present disclosure provides a method for stabilizing at least one of fHBP A and Neisseria adhesin A (NadA) proteins in an immunogenic composition, the method comprising at least: a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AlPO4 adjuvant selected in step a) with fHBP A or NadA protein; c) obtaining an immunogenic composition in which the fHBP A or NadA protein is stabilized; The present invention relates to a method comprising:

[0577] This method makes it possible to obtain immunogenic compositions in which fHBP A and / or NadA are stabilized.

[0578] The methods of the present disclosure may further comprise the step of adding at least one antigen.

[0579] For compositions comprising NadA protein, the at least one further antigen may be selected from fHBP A, fHBP B and dOMV.

[0580] For compositions comprising fHBP A, the at least one further antigen may be selected from fHBP B, NadA protein and dOMV.

[0581] The at least one additional antigen may be added before or after the step of combining AlPO4 with the fHBP A or NadA protein.

[0582] The additional antigens may be added to the fHBP A or NadA protein in separate steps, or may be combined and then added in a single step, in which case they may be added as a partial combination of antigens.

[0583] The step or steps of adding additional antigens may occur before or after combining AlPO4 with the fHBP A or NadA protein.

[0584] In some embodiments, step b) comprises combining an AlPO4 adjuvant with a combination of fHBP A, fHBP B, NadA protein and dOMV, wherein the combining is performed in any order.

[0585] The fHBP A and B, NadA protein and dOMV may be as detailed above.

[0586] The method may also include the step of adding a combination of MenACWY polysaccharides conjugated to a protein carrier as detailed above.

[0587] The stability of antigens in the compositions of the present disclosure can be assayed by methods well known in the art, including measuring the light scattering of a sample, the 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 by differential scanning calorimetry (DSC).

[0588] For example, the stability of an antigen such as NadA or fHBP A in a composition of the present disclosure may be determined by subjecting the antigen under consideration to heat stress by incubating the immunogenic composition at 45°C and measuring the antigenicity as a function of time, for example at 0, 7, 14 and 28 days.

[0589] The antigen in the compositions of the present disclosure may maintain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of its relative antigenicity compared to a reference standard, e.g., the antigenicity measured at TO (i.e., the day of formulation or the day storage conditions were changed), 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 at a temperature ranging from 4°C to 8°C.

[0590] Combining the AlPO4 adjuvant of the present disclosure with fHBP A and B is carried out under conditions suitable to achieve adsorption of fHBP A and B to the AlPO4.

[0591] The % of fHBP A and B adsorbed is as indicated above.

[0592] fHBP A and B may each be adsorbed onto AlPO in an amount less than 85% of the total amount of fHBP B present in the composition, or in an amount ranging from about 50% to about 85% or less of the total amount of fHBP A or fHBP B, respectively, present in the composition.

[0593] Suitable conditions to ensure adsorption of fHBP A and B to AlPO4 include pH, temperature, and PZC of AlPO4. These conditions may be as indicated above.

[0594] For example, the pH may be in the range of 5.5 to 7.0.

[0595] The temperature may range from 4°C to 25°C.

[0596] Compositions of the present disclosure may be stored at a temperature ranging from about 4° C. to about 25° C. For example, compositions of the present disclosure may be stored at a temperature of about 4° C. or about 8° C. or about 4° C.

[0597] The present disclosure provides a method for the preparation of a serogroup B antibody comprising at least one N. meningitidi serogroup B antigen and having a settling time (T) ranging from about 3.5 minutes to about 10 minutes. onset ), comprising at least a) selecting an AlPO4 adjuvant with a PZC below 5; b) combining the AlPO4 adjuvant selected in step a) with the at least one N. meningitidi serogroup B antigen; c) obtaining said immunogenic composition; The present invention relates to a method comprising:

[0598] The at least one antigen may be fHBP A, fHBP B, NadA protein or dOMV, and combinations thereof.

[0599] Settling start time (T onset ) can range from about 4 minutes to about 9 minutes or from about 4.5 minutes to about 8.5 minutes.

[0600] Advantageously, a settling onset time of at least 3.5 minutes or greater ensures that the components of the composition will remain in suspension during manufacturing, thus allowing for more consistent manufacturing.

[0601] Additional antigens, such as MenACWY polysaccharide conjugated to a protein carrier, may be mixed with the compositions as disclosed herein. The additional antigens may be added to the immunogenic compositions of the present disclosure just prior to administration to a patient, optionally by means of a dual-chamber syringe that mixes the composition of the present disclosure with at least the additional antigen prior to administration.

[0602] The manufacturing methods of the present disclosure can be used to manufacture vaccines.

[0603] Use and Method The present disclosure relates to an immunogenic composition as disclosed herein for use as a medicament, in particular as a vaccine.

[0604] The immunogenic compositions of the present disclosure or vaccines comprising the immunogenic compositions of the present disclosure may be for use in methods of protecting against meningococcal infection, which may be caused by N. meningitidis serogroup B strains.

[0605] The immunogenic compositions or vaccines of the present disclosure may be for use in methods of inducing an immune response against N. meningitidis serogroup B strains.

[0606] The immunogenic composition or vaccine of the present disclosure may be for use in a method of protecting an individual from meningococcal infection, the method comprising at least administering the immunogenic composition to the individual.

[0607] The present disclosure relates to a method of protecting an individual from meningococcal infection, comprising at least administering to the individual the immunogenic composition or a vaccine comprising the immunogenic composition.

[0608] The immunogenic composition or vaccine of the present disclosure may be for use in a method of reducing the risk of an individual developing invasive meningococcal disease caused by meningococcal infection, the method comprising at least the step of administering the immunogenic composition or vaccine to an individual.

[0609] The present disclosure relates to a method for reducing the risk of an individual developing invasive meningococcal disease caused by meningococcal infection, the method comprising at least the step of administering to the individual an immunogenic composition or vaccine of the present disclosure.

[0610] The immunogenic compositions or vaccines of the present disclosure may be for use in a method of eliciting an immune response to N. meningitidis serogroup B strains in an individual, the method comprising at least the step of administering the immunogenic composition or vaccine to the individual.

[0611] The present disclosure relates to a method of eliciting an immune response against N. meningitidis serogroup B strains in an individual, the method comprising at least the step of administering to the individual an immunogenic composition or vaccine of the present disclosure.

[0612] The meningococcal infection may be a N. meningitidis serogroup B infection. The immunogenic composition may be a vaccine.

[0613] The present disclosure relates to the use of an AlPO adjuvant having a PZC of less than 5 to enhance the immune response induced by a composition comprising a N. meningitidis fHBP B antigen against a N. meningitidis serogroup B strain expressing a fHBP B heterologous to the fHBP B antigen of the composition.

[0614] The present disclosure relates to an AlPO4 adjuvant having a PZC of less than 5 in an immunogenic composition comprising a N. meningitidis fHBP B antigen, for use in a method of enhancing the immune response induced by the composition against a N. meningitidis serogroup B strain that expresses an fHBP B heterologous to the fHBP B antigen of the composition.

[0615] According to another of its objects, the present disclosure relates to the use of an AlPO adjuvant having a PZC of less than 5 to enhance the immune response induced by a composition comprising a N. meningitidis fHBP B antigen against a N. meningitidis serogroup B strain expressing an fHBP B homologous to the fHBP B antigen of said composition.

[0616] According to another of its objects, the present disclosure relates to the use of an AlPO adjuvant having a PZC of less than 5 to enhance the immune response induced by a composition comprising a N. meningitidis fHBP A antigen against a N. meningitidis serogroup B strain expressing a fHBP A heterologous to the fHBP A antigen of said composition.

[0617] According to another of its objects, the present disclosure relates to the use of an AlPO adjuvant having a PZC of less than 5 to enhance the immune response induced by a composition comprising a N. meningitidis fHBP A antigen against a N. meningitidis serogroup B strain expressing an fHBP A homologous to the fHBP A antigen of said composition.

[0618] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC below 5 to stabilize at least one fHBP A in an immunogenic composition.

[0619] The present disclosure relates to the use of an AlPO4 adjuvant with a PZC below 5 to stabilize at least one Neisserial adhesin A (NadA) antigen in an immunogenic composition.

[0620] The present disclosure provides a method for determining the time to sedimentation (T) of a composition comprising at least one Neiseria meningitidis serogroup B antigen. onset ) in the range of about 3.5 minutes to about 10 minutes, or in the range of about 4 minutes to about 9 minutes, or in the range of about 4.5 to about 8.5 minutes.

[0621] The at least one Neiseria meningitidis serogroup B antigen may be from the group including fHBP A, fHBP B, NadA protein, dOMV, and combinations thereof.

[0622] The at least one Neiseria meningitidis serogroup B antigen can be a combination of fHBP A and fHBP B.

[0623] The present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 to adjuvant 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.

[0624] The present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 to prepare 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.

[0625] The present disclosure relates to a method of inducing an immune response against a Neisseria meningitidis serogroup B strain in an individual in need thereof, the method comprising administering to the individual at least an immunogenic composition according to the present disclosure, wherein the administering step induces an immune response against the Neisseria meningitidis serogroup B strain.

[0626] The present disclosure relates to a method of enhancing an immune response induced by a composition comprising a N. meningitidis fHBP B antigen against a N. 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 administering at least an immunogenic composition according to the present disclosure to the individual, wherein the administering step induces an enhanced immune response against the heterologous N. meningitidis serogroup B strain.

[0627] The individual in accordance with the methods and uses of the present disclosure may be a mammal, such as a human, and may be, for example, an infant, a toddler, a child, a teenager, a young adult, an adult, or an elderly person. In one embodiment, the individual may be 6 weeks old, 2 months old, or 10 years old or older. In exemplary embodiments, the individual may be 6 weeks old to 55 years old or older, such as 2 months old to 55 years old or older, or for example, 10 years old to 55 years old or older.

[0628] The methods generally involve administering an effective amount of a subject immunogenic composition to an individual in need thereof. Amounts effective for therapeutic use will depend, for example, on the antigenic composition, the method of administration, the patient's weight and general health, and the judgment of the prescribing physician. Single or multiple doses of the antigenic composition can be administered, depending on the dosage and frequency and route of administration required and tolerated by the patient.

[0629] The immunogenic compositions as disclosed herein may be administered in a 2, 3, 2+1 or 3+1 dose regimen.

[0630] In one embodiment, the immunogenic compositions disclosed herein may be administered in two or three doses. Subsequent doses may be administered about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 months after the previous dose. In one embodiment, subsequent doses may be administered about 1, about 2, about 5, about 6, about 8, about 10, about 12, about 14, or about 16 months after the previous dose. In one embodiment, subsequent doses may be administered about 1, about 2, about 5, about 6, or about 8 months after the previous dose. In one embodiment, subsequent doses may be administered about 30 days, about 60 days, or about 180 days after the previous dose.

[0631] In a two-dose regimen, the second dose can be administered about one month after the first dose, or about two months after the first dose, or about six 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.

[0632] In a two-dose regimen, the second dose may be administered about two months after the first dose. Alternatively, in a two-dose regimen, the second dose may be administered about 60 days after the first dose. Such a two-dose regimen may be suitable for young children.

[0633] In a three-dose regimen, the second dose can be administered about one month after the first dose, and the third dose can be administered about six months after the first dose. Alternatively, in a three-dose regimen, the second dose can be administered about 30 days after the first dose, and the third dose can be administered about 180 days after the first dose. Such a three-dose regimen can be suitable for adults and / or adolescents.

[0634] In a three-dose regimen, the second dose can be administered about 2 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.

[0635] In one embodiment, two or three doses may be administered followed by a third or fourth dose. This subsequent dose may be administered at least one year after the last of the two or three doses, for example, 16 months after the last dose. In such a regimen, the first two or three doses may qualify as prime doses, and the subsequent one (+1) may qualify as a boost dose.

[0636] In one embodiment, infants and young children, e.g., 6 weeks or 2 months to 2 years of age, may receive a 2+1 or 3+1 dose regimen. In another embodiment, children, e.g., 2-10 years of age, may receive a 2 dose regimen. In another embodiment, teenagers and adults, e.g., 10-55 years of age, may receive a 2+1 dose regimen.

[0637] The immunogenic compositions as disclosed herein may be administered by any suitable route. For example, administration by the intramuscular route may be considered. [Example]

[0638] The following examples describe the embodiments of the present disclosure that are currently best known. However, it is to be understood that the following are merely exemplary or illustrative of the application of the principles of the present disclosure. Many variations and alternative compositions, methods, and systems can be envisioned by those skilled in the art without departing from the spirit and scope of the present disclosure.

[0639] Example 1: Materials and Methods Formulation of AlPO4 with a low point of zero charge (mod-AlPO4 adjuvant) Aluminum phosphate adjuvant gels with PZCs ranging from 5 to 7 were titrated with phosphate buffer / salt solutions, allowing the phosphate groups from the buffer or salt solution to exchange with hydroxyl groups on the surface of the aluminum phosphate adjuvant. The PZC of AlPO4 can be lowered by using any orthophosphate or phosphate-donating salt or buffer solution.

[0640] A variety of different methods can be used to obtain AlPO4 with the desired PZC.

[0641] In one method, AlPO4 of different PZCs was made by adding phosphate buffer pH 5.8 formulated by combining 0.5 M monobasic sodium phosphate and 0.5 M dibasic sodium phosphate.

[0642] In the second method, the PZC of AlPO4 was modified by titrating AlPO4 with a 0.5 M stock solution of monobasic sodium phosphate.

[0643] AlPO4 adjuvants with a PZC below 5 are modified AlPO4 adjuvants and are referred to hereafter in this Example as "mod-AlPO4 adjuvants." To prepare 100 mL of modified AlPO4, 80 mL of adjuvant (mod-AlPO4 adjuvant) was combined with 20 mL of 0.5 M sodium phosphate buffer, pH 5.8, at room temperature and stirred for at least 30 minutes. The modified AlPO4 (mod.AlPO4 or mod-AlPO4) was stored at 2-8°C until use.

[0644] AlPO4 adjuvants with a PZC greater than 5 are unmodified AlPO4 adjuvants and will be referred to hereafter in this Examples section as "AlPO4 adjuvants."

[0645] In the following studies, the mod-AlPO4 adjuvant was modified as described above to set the PZC at about 4.5, while the AlPO4 adjuvant was set to have a PZC of about 5.2.

[0646] Preparation of MenB immunogenic composition (MenB) Non-lipidated mutant fHBP A05 (A05tmN) To prepare non-lipidated A05tmN, three point mutations (G220S, L130R, and G133D, numbered based on SEQ ID NO: 6) were introduced into the wild-type fHBP A05 sequence. In addition, the lipidable cysteine residue at the N-terminus was replaced with a methionine residue (non-lipidated A05tmN: SEQ ID NO: 8). A DNA sequence encoding the A05tmN antigen was synthesized and then cloned into a plasmid construct. Briefly, DNA sequences for Xba1 and Xho1 sites were added to both ends of the A05tmN sequence. To generate an expression plasmid, the Xba1 / Xho1-containing pET28a(+) plasmid was digested. The DNA sequence encoding A05tmN with Xba1 and Xho1 sites was ligated into Xba1 / Xho1-digested pET28a(+) 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 a cell bank was produced after three rounds of colony purification.

[0647] The A05tmN expression construct was transformed into Escherichia coli (E. coli) strain and amplified in semi-defined medium (pH 6.8, dissolved oxygen: 20%) at 37°C under agitation. Antigen expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0648] Cultures were harvested as raw bulk, and the bacterial biomass was separated from the medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was homogenized to produce a cell homogenate. The homogenate was then centrifuged to collect the pellet fraction. The homogenate pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5) and subjected to a pH shock treatment (pH 12, 1 hour at room temperature with mixing). The pH was returned to 8.5 with 85% phosphoric acid. The supernatant fraction of the pH-shocked material was collected after centrifugation and then filtered to obtain the filtered supernatant.

[0649] The supernatant was conditioned to pH 8.5 and a conductivity of less than 5.0 mS / cm and loaded onto a GigaCap Q-650M capture column. The elution pool was conditioned to 0.9 M ammonium sulfate (AmS) and then further purified by intermediate chromatography on Toyopearl Phenyl 600M. After hydrophobic interaction chromatography, the elution pool was conditioned to pH 8.5 and a conductivity of less than 8.0 mS / cm and further purified by Nuvia aPrime 4A chromatography. This was followed by a final ultrafiltration and diafiltration using a 5 kDa regenerated cellulose tangential flow filtration (TFF) membrane and 0.2 μm filtration.

[0650] Non-lipidated mutant fHBP B01 (B01smN) To prepare non-lipidated B01smN, a single point mutation (H248L, numbering based on SEQ ID NO: 6) was introduced into the wild-type fHBP B01 sequence. In addition, the lipidable cysteine residue at the N-terminus was replaced with a methionine (non-lipidated B01smN: SEQ ID NO: 9). The DNA sequence of B01smN was synthesized and then cloned into a plasmid construct. Briefly, DNA sequences for Xba1 and Xho1 sites were added to both ends of the B01smN sequence. To generate an expression plasmid, the Xba1 / Xho1-containing pET28a(+) plasmid was digested. The DNA sequence encoding B01smN with Xba1 and Xho1 sites was ligated into Xba1 / Xho1-digested pET28a(+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba1 / Xho1 digestion. The B01smN plasmid was transformed into E. coli and a cell bank was produced after three rounds of colony purification.

[0651] The B01smN expression construct was transformed into Escherichia coli (E. coli) strain and amplified in semi-defined medium (pH 6.8, dissolved oxygen: 20%) at 37°C under agitation. Antigen expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0652] Cultures were harvested as raw bulk, and the bacterial biomass was separated from the medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was homogenized to produce a cell homogenate. The homogenate was then centrifuged and the supernatant fraction was collected. The supernatant fraction was then filtered.

[0653] The filtered supernatant was conditioned to pH 8.5 and a conductivity of less than 5.0 mS / cm and loaded onto a CaptoQ ImpRes column for purification in bind-and-elute mode. The CaptoQ ImpRes elution pool was then conditioned to 1.8 M AmS and loaded onto a second column, Phenyl Sepharose HP. After elution, the material was concentrated and diafiltered with acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0) using a 5 kDa Ultracel TFF membrane, followed by 0.2 μm filtration.

[0654] NadA A truncated version of NadA was prepared from NadA_MC58. The truncated NadA lacks the leader sequence (residues 1-23) and anchor domain (residues 308-362) of NadA_MC58 (truncated NadA: SEQ ID NO: 5). In this truncated NadA_MC58 sequence, the first amino acid after the leader sequence is an alanine, which was replaced with a methionine. The DNA sequence encoding the truncated NadA was synthesized and then cloned into a plasmid construct. DNA sequences for Xba1 and Xho1 sites were added to both ends of the NadA sequence. To generate the expression plasmid, the Xba1 / Xho1-containing pET28a(+) plasmid was digested. The DNA sequence encoding NadA with the Xba1 and Xho1 sites was ligated into Xba1 / Xho1-digested pET28a(+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba1 / Xho1 digestion. The NadA plasmid was transformed into E. coli and a cell bank was produced after three rounds of colony purification.

[0655] The NadA1-transformed E. coli strain was grown in semi-defined medium (pH 6.8, dissolved oxygen: 20%) at 37°C under agitation. Antigen expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0656] Cultures were harvested as raw bulk, and the bacterial biomass was separated from the medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was homogenized to produce a cell homogenate. The homogenate was then centrifuged and the supernatant fraction was collected. The supernatant fraction was then filtered.

[0657] The supernatant fraction was loaded onto a Capto DEAE column. The Capto DEAE elution fraction was conditioned with powdered AmS until a concentration of 500 mM AmS was achieved. The conditioned Capto DEAE elution fraction was loaded onto a Toyopearl Butyl-650M column. The Toyopearl Butyl-650M elution fraction was loaded onto a CHT Type I 40 μm column, and the CHT elution fraction was concentrated using a 30 kDa regenerated cellulose TFF membrane, followed by diafiltration with 50 mM sodium acetate, 150 mM NaCl, pH 6.0. Following TFF, the product was filtered through a 0.2 μm filter to produce the NadA antigen.

[0658] dOMV dOMVs were purified from wild-type N. meningitidis (Nm) serogroup B strain 99M, provided by the Walter Reed Army Institute of Research (WRAIR).

[0659] Nm B 99M was cultured in the chemically defined medium described by Fu et al. (Biotechnology (NY). 1995 Feb;13(2):170-4) and U.S. Pat. No. 5,494,808 in the presence of 1 g / L yeast extract and 1M Hepes at 37°C under 5% CO2.

[0660] Culture harvesting was performed using low-speed centrifugation of the heat-treated suspension (55°C for 2 hours) to recover the moist bacterial pellet. dOMVs were extracted from the bacterial outer membrane and depleted of lipooligosaccharides by two consecutive detergent-mediated extraction steps (56°C for 15 minutes) with an extraction buffer composed of a detergent (sodium deoxycholate) (as disclosed in Helting et al., Acta Pathol Microbiol Scand C. 1981 Apr;89(2):69-78). Sodium deoxycholate and EDTA solubilized the bacterial outer membrane, which then reassembled into dOMVs (vesicles and particulate matter). Resuspension was completed by homogenizing the pellet suspended in the extraction buffer using an Ultra-Turrax (rotor-stator arrangement). The dOMV supernatants were pooled and then treated with benzonase in the presence of MgCl2 (37°C for 15 minutes).

[0661] Following dOMV extraction, the dOMVs were concentrated using 300 kDa modified polyethersulfone (mPES) hollow fibers. Several ultracentrifugation steps were used to separate the dOMVs from "soluble" contents, such as nucleic acids, cytoplasmic proteins, extracted lipopolysaccharides, or buffer components. The resulting pellet was then resuspended in extraction buffer using an Ultra-Turrax (rotor-stator arrangement) at minimum speed for several seconds. After primary resuspension, high-pressure homogenization was used to thoroughly resuspend the dOMVs in extraction buffer, increasing the amount of detergent contact with the dOMV surface.

[0662] After centrifugation, the supernatant was finally filtered through a 0.45 / 0.2 μm cellulose acetate filter, and the dOMVs were recovered in water for injection (WFI).

[0663] MenB immunogenic composition The MenB antigens in the MenB immunogenic composition were purified non-lipidated mutant A05 fHBP (A05tmN), non-lipidated mutant fHBP (B01smN), NadA, and dOMV. The A05tmN, B01smN, NadA, and dOMV antigens were combined with aluminum phosphate adjuvant (AlPO4) (PZC5.2) or mod-AlPO4 adjuvant (PZC4.5).

[0664] The medium consisted of acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0).

[0665] The AlPO4 adjuvant was either unmodified AlPO4 adjuvant (PZC5.2; 1.00 mg Al / mL) or mod-AlPO4 adjuvant (PZC4.5; 1.00 mg Al / mL) prepared as indicated above.

[0666] To formulate the immunogenic composition formulations, unmodified (PZC5.2) 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 blended 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 AlPO4).

[0667] For stability data and PZC comparisons (4.5 and 4.8), a final aluminum concentration of 0.8 mg Al / mL AlPO4 was maintained. Adsorbed antigen is measured by determining the amount of unadsorbed antigen using RP-HPLC for MenB antigen and HPAEC-PAD for ACYW conjugates.

[0668] The estimated amount of residual phosphate buffer in the final composition is approximately 24 mM.

[0669] Preparation of MenACWY and MenPenta immunogenic compositions Preparation of MenACWY immunogenic composition The MenACWY immunogenic composition is obtained from MENQUADFI®, a commercially available vaccine containing ACWY polysaccharide antigens derived from and conjugated to tetanus toxoid (TT), as disclosed in WO 2018 / 045286 A1. This formulation contains N. meningitidis capsular polysaccharides from serogroups A, C, Y, and W135 individually conjugated to tetanus toxoid protein. The target active ingredient concentration is 10 μg of each polysaccharide and approximately 55 μg of tetanus toxoid protein per 0.5 mL dose. These antigens were 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).

[0670] Preparation of MenPenta Immunogenic Composition (MenPenta) MenPenta formulations were prepared by combining two nonlipidated factor H binding proteins (fHBPs) from subgroups A and B targeting N. meningitidis B strains, Neisserial adhesin A (NadA), and detergent-extracted outer membrane protein vesicles (dOMVs), prepared as indicated above, with serogroup polysaccharides A, C, Y, and W135 conjugated to tetanus toxoid as a carrier, obtained as indicated above, with either (i) aluminum phosphate adjuvant (AlPO4) (PZC5.2) or (ii) mod-AlPO4 adjuvant (PZC4.5).

[0671] The preparation was mixed by stirring at ambient temperature for at least 30 minutes and stored at 2-8°C until use.

[0672] Settling start time Sedimentation tests were performed using a TURBISCAN LAB™. A 1.5 mL volume of the composition to be assayed was filled into a 4 mL glass tube and placed on an adapter corresponding to the vial size. Samples of MenB or MenPenta compositions containing AlPO4 with a PZC of 5.2 or 4.5 (obtained as indicated above) were monitored for transmission and backscattering every 25-30 seconds for 30 minutes. Both transmission and backscattering were measured. Measurements were performed by scanning from the bottom to the top of the measurement cell. The measurement temperature was set at 28°C.

[0673] Pyrogenicity of immunogenic composition (IL-6 EC 50 (measurement of The immunogenic compositions tested (MenB in AlPO4PZC5.2 or mod-AlPO4PZC4.5 - prepared as indicated above) were assayed for pyrogenicity using the Monocyte Activation Assay (MAT).

[0674] The MAT is based on the ability of human monocyte cells to secrete endogenous pyrogens (pro-inflammatory cytokines) in response to the detection of exogenous pyrogens in a test sample. The monocyte activation test (MAT) works by predicting the human response to pyrogens in terms of the same endogenous cytokines produced during human fever. For test samples with inherent pyrogens (e.g., lipoproteins, lipooligosaccharides, other unknown components), the MAT can quantify the intrinsic pyrogenicity and is therefore used to demonstrate consistency.

[0675] MAT is based on a pool of peripheral blood mononuclear cells (PBMCs) from eight different healthy human donors as the monocytic cell source and human interleukin-6 (IL-6) as a readout.

[0676] In this study, the goal was to compare the pyrogenicity of immunogenic compositions formulated with either AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC4.5). All samples were adjusted to the same protein content, serially diluted, and plated with human PBMCs in 96-microwell plates for overnight incubation. After incubation, the supernatants were transferred to 96-microwell plates, and immunodetection of IL-6 was performed by homogeneous time-resolved fluorescence (HTRF). The half-effective concentration (EC) was calculated using the 4PL extrapolation model of the dose-response curve. 50 ) is the parameter used to compare the intrinsic pyrogenicity of each composition. The minimum value is EC 50 and the highest value is the intrinsic pyrogenicity of the product tested.

[0677] Measurement of antigen adsorption to aluminum The adsorption rate of the antigen to mod-AlPO4 or AlPO4 was measured by determining the relative concentration of the unadsorbed antigen in the supernatant after centrifugation of the aluminum adjuvant-bound antigen using the following formula:

number

[0678] The concentrations of A05tmN, B01smN, NadA, and dOMV were determined by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0679] The reversed-phase liquid chromatography (RP-LC) method from Nompari et al. Talanta 178 (2018) 552-562 was performed on an Agilent 1260 Infinity HPLC instrument with ultraviolet detection.

[0680] The antigen content of the supernatant and the desorbed (bound) fraction were determined to determine the % adsorption.

[0681] Supernatant samples were prepared by centrifuging the adsorbed samples at 3000 rcf for 5 minutes at 22°C. An appropriate volume of supernatant was mixed with the detergent Zwittergent 3-14 to achieve a final concentration of 0.1% w / v Zwittergent 3-14 in the sample at the lowest sample dilution (approximately 0.95x). The samples were then heated at 60°C for 1 hour with shaking at 300 rpm in a microtube warmer. To prepare all samples, antigen desorption was achieved by treating the entire adsorbed sample (total volume) with 5% w / v citrate, aluminum chelator, and 0.1% Zwittergent 3-14. All samples and standards were heated at 60°C for 1 hour with shaking at 300 rpm, centrifuged at 3000 rcf for 5 minutes at 22°C, and then stored at 10°C in an HPLC autosampler until HPLC analysis.

[0682] On the day of testing, reference standard working solutions were freshly prepared by diluting the antigen reference standards A05tmN, B01smN, and NadA to the appropriate concentrations in 150 mM sodium acetate + 50 mM sodium chloride, pH 6.3 buffer containing 0.1% Zwittergent 3-14. NadA was used as a heterologous reference standard to quantify dOMV protein components.

[0683] Optimal separation of all antigens was achieved using a Waters BioResolve RP mAb Polyphenyl column, 450 Å, 2.7 μm, 2.1 mm × 150 mm. Liquid chromatography-mass spectrometry (LC-MS) grade 0.1% TFA in water and 0.1% TFA in ACN were used as the aqueous and organic mobile phases, respectively. The chromatographic gradient started with 10% organic phase and ended with 80% organic phase, and the column temperature was 70°C. The detection wavelength used was 215 nm. Protein concentrations were determined by interpolating the amount of each antigen in nanograms (ng) from the appropriate calibration curve and then dividing by the injection volume (in μL) to obtain ng / μL or μg / mL.

[0684] The concentrations of tetanus toxoid protein-conjugated N. meningitidis capsular polysaccharides of serogroups A, C, Y, and W135 were determined by high-performance anion-exchange chromatography / pulsed amperometric detection (HPAEC-PAD).

[0685] Quantification of polysaccharide content and % adsorption to AlPO4 in drug product vaccines was determined by preparing samples, acid hydrolysis to generate monosaccharides, and chromatographic analysis of individual monosaccharides by HPAEC-PAD.

[0686] Desorbed samples were prepared by centrifuging aliquots of drug product samples at 14,000 rpm at room temperature for 30 minutes. The supernatant was removed and diluted within the standard curve range using sodium acetate / sodium chloride buffer. An aliquot of the drug product sample (adsorbed) was also diluted within the standard curve range using sodium acetate / sodium chloride buffer. The diluted adsorbed drug product reference standard, supernatant, and polysaccharide were prepared as sample sets.

[0687] Hydrolysis and chromatographic conditioning were performed as described by Gudlavalleti et al. (Anal Chem. 2014 Jun 3;86(11):5383-90. doi:10.1021 / ac5003933. Epub 2014 May 20. PMID:24810004.).

[0688] Calibration curves were calculated using linear regression from tetravalent polysaccharide-based reference standards for serogroups A, C, W, and Y. Polysaccharide concentrations for both adsorbed and desorbed (supernatant) samples of A, C, W, and Y were interpolated from their respective curves. % adsorption was calculated as desorbed / drug product sample × 100.

[0689] Determination of the immunogenicity of MenB immunogenic compositions formulated with mod-AlPO4 adjuvant (PZC4.5) or AlPO4 adjuvant (PZC5.2) Sample

[0690] [Table 1]

[0691] Dose-Timing Regimen Three groups (A, B, and C) of eight rabbits (female; strain: NZW KBL; 9-10 weeks old on 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 either AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC4.5), respectively. These formulations were administered by intramuscular (IM) injection on D0 and D28 (500 μL into the right thigh for the first injection and 500 μL into the left thigh for the second injection). On D0, D28, and D42 (2 weeks after the final injection), blood samples were collected from the median ear artery of the rabbits under local anesthesia for all groups.

[0692] [Table 2]

[0693] Biosample collection and hSBA Biological sample collection On D0 and D42, blood samples were collected from the median ear artery under local anesthesia. Local anesthesia was achieved by applying an anesthetic cream (Elma®) to the rabbit's ears 5 minutes before blood samples were collected. Rabbits were also maintained under chemical anesthesia with Rompun and Imalgene products.

[0694] Blood samples were collected into tubes containing a clot activator and serum separator (BD Microtainer SST 5mL, reference number 15388989). The tubes were centrifuged at 3500 rpm for 15 minutes to separate serum from blood cells. Serum was transferred to deepwell (ritter) plates and heat-inactivated at 56°C for 30 minutes. They were stored at -20°C until use in IgG purification and bacterial killing assays.

[0695] hSBA IgG purification of rabbit serum for hSBA testing Purification of IgG was necessary to avoid nonspecific bactericidal killing induced by rabbit sera collected on D0 and D42.

[0696] Purification of rabbit serum was performed using rProtein A GravtiTrap™ columns (GE healthcare GE28-9852-54) and Ab Buffer Kit (GE Healthcare ref 28-9030-59).

[0697] First, the column was equilibrated with binding buffer (20 mM sodium phosphate, pH = 7). After adjusting the pH of the serum to 7 with binding buffer (V / V), serum was added to the column for IgG binding. The column was washed with binding buffer. Next, elution buffer (glycine HCl 0.1 M pH 2.7) was added to the column to collect the IgG. To preserve the activity of the IgG, neutralization buffer (Tris-HCl 1 M, pH 9.0) was added to the eluted fraction to achieve a final pH of approximately 7. Quantification of the IgG concentration was performed using a Nanodrop.

[0698] Serum bactericidal activity 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 Neisseria meningitidis serogroup B (i.e., serum bactericidal activity - SBA). This assay was performed in the presence of human complement (hSBA). In the presence of complement and several immunoglobulin classes, lytic antigen-antibody complexes are formed on the surface of target bacteria, causing their death. Serum SBA levels can be determined by observing the bactericidal effect resulting from target-specific antibodies present in the serum.

[0699] The bactericidal titer is the dilution at which ≥ 50% occurs. The resulting number of bacterial colonies present in the well is inversely proportional to the level of functional antibodies present in the serum, which is directly proportional to the immune response of the animal or human subject.

[0700] The SBA assay measures the ability of antibodies to lyse bacteria in the presence of complement. The bactericidal titer of a serum is defined as the reciprocal of the highest dilution of the test serum that produces at least 50% killing compared to complement control wells containing no serum.

[0701] The resulting number of bacterial colonies present in the well is inversely proportional to the level of functional antibodies present in the serum, which is directly proportional to the immune response of the animal or human subject.

[0702] The source of complement was human complement (Ig-depleted human serum). Briefly, serum was heat-inactivated at 56°C for 30 min and then incubated in a 96-well microplate with Ca. 2+ and Mg 2+ and Dulbecco's PBS buffer containing 0.2% gelatin or Ca 2+ / Mg 2+ Two-fold serial dilutions (9 times) are made in Dulbecco's PBS buffer containing 0.1% dextrose and 0.5% bovine serum albumin.

[0703] All sera evaluated in the SBA assay are heat inactivated in a 56°C water bath for 30 minutes to inactivate endogenous complement activity.

[0704] Bacterial pre-culture was performed on Mueller-Hinton agar (Petri dishes) for 18 hours at 37°C in 5% CO2 to achieve confluent bacterial growth.

[0705] Bacteria were then grown in brain heart infusion (BHI) medium (for strain no. 6, supplemented with 4-HPA 5 mM to induce NadA expression - see Table 3) at +37°C for 2 hours and 30 minutes with shaking (100 rpm).

[0706] Meningococcus bacteria 1.4 10 4The working bacterial suspension was diluted to reach CFU / mL. 25 μL of the working bacterial suspension, 50 μL of prediluted serum, and 25 μL of diluted human complement (final concentration 15%) were placed in a 96-well microplate and incubated at +37°C for 1 hour with shaking (100 rpm). The Zephyr robotic application automatically dispensed 40 μL into each well of a square plate (40 x 40) containing Mueller-Hinton agar. The agar plate was incubated at +37°C with 5% CO2 for 12 ± 4 hours.

[0707] After incubation, the number of colonies per well was counted using Microvision's Cybele software.

[0708] 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 into the Sanofi Universal Exporter (SUE) by selecting the SBA WARP module.

[0709] Neisseria meningitidis serogroup B strains for SBA determination

[0710] [Table 3]

[0711] Data analysis ANOVA analysis was performed with test product as a fixed factor.

[0712] Determination of the immunogenicity of MenACWY or MenPenta immunogenic compositions formulated with mod-AlPO4 adjuvant (PZC4.5) Sample

[0713] [Table 4]

[0714] Dose-Timing Regimen Three groups (1, 2, and 3) of six rabbits (female; strain: NZW KBL; 9-10 weeks old on D0) were used. Group 1 received the MenACWY immunogenic composition without AlPO4, while groups 2 and 3 received the MenACWY immunogenic composition containing 400 μg of mod-AlPO4 (PZC4.5) adjuvant either without or with the MenB immunogenic composition, respectively. These formulations were administered by IM injection on D0 and D28 (500 μL into the right thigh for the first injection and 500 μL into the left thigh for the second injection). Blood samples were collected from the median ear artery of the rabbits under local anesthesia on D0, D28, and D42 (2 weeks after the final injection on D42 for all groups).

[0715] [Table 5]

[0716] [Table 6]

[0717] Biosample collection and hSBA Biological sample collection Biological sampling was performed on D0, D28 and D42 as per the procedures described above for determining the immunogenicity of the MenB immunogenic compositions.

[0718] hSBA of MenB antigen An hSBA of MenB antigen was performed as described above for determining the immunogenicity of the MenB immunogenic composition.

[0719] hSBA of MenACWY antigen IgG purification of rabbit serum for hSBA testing Purification of IgG was necessary to avoid nonspecific bactericidal killing induced by rabbit sera collected on D0 and D42.

[0720] Purification of rabbit serum was performed using rProtein A GravtiTrap™ columns (GE healthcare GE28-9852-54) and Ab Buffer Kit (GE Healthcare ref 28-9030-59).

[0721] First, the column was equilibrated with binding buffer (20 mM sodium phosphate, pH = 7). After adjusting the pH of the serum to 7 with binding buffer (V / V), serum was added to the column for IgG binding. The column was washed with binding buffer. Next, elution buffer (glycine HCl 0.1 M pH 2.7) was added to the column to collect the IgG. To preserve the activity of the IgG, neutralization buffer (Tris-HCl 1 M, pH 9.0) was added to the eluted fraction to achieve a final pH of approximately 7. Quantification of the IgG concentration was performed using a Nanodrop.

[0722] Serum bactericidal activity 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.

[0723] The source of complement was human complement (Ig-depleted human serum). Briefly, serum was heat-inactivated at 56°C for 30 min and then incubated in a 96-well microplate with Ca. 2+ / Mg 2+ Two-fold serial dilutions (9 times) were made in Dulbecco's PBS buffer containing 0.1% dextrose and 0.5% bovine serum albumin (dilution buffer).

[0724] Bacterial precultures were grown on PVX medium plates (chocolate agar + PolyViteX) at +37°C and 5% CO2 for 15 hours for serogroup Y or 18 hours for serogroups A, C, and W-135 to obtain isolated colonies. Bacteria from overnight plates were spread onto fresh PVX medium plates to obtain a thin veil of confluent bacterial growth after 4 hours at +37°C and 5% CO2. After incubation, bacteria were diluted to 8.10 3 CFU / mL was achieved. 50 μL of prediluted 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 minutes for serogroups C, W-135, and Y or 90 minutes for serogroup A. After the appropriate incubation period, 50 μL of each well was transferred to a flat-bottom plate, and 100 μL of TSB agar was added to all wells. Plates were incubated at +37°C with 5% CO2 for 6-8 hours.

[0725] After incubation, the number of colonies per well was counted using a Cytation 7 instrument and Gen5 software (Biotek).

[0726] 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 into the Sanofi Universal Exporter (SUE) by selecting the Gen5 WARP module.

[0727] Data analysis A two-way analysis of variance (ANOVA) was performed with time, test substance, and their interaction as fixed factors. Data were paired by time.

[0728] Determination of antigen stability in MenB, MenACWY, and MenPenta (MenB+MenACWY) immunogenic compositions Measurement of antigenicity of MenB antigen Antigen stability under heat stress was measured 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.

[0729] A direct enzyme-linked immunosorbent assay (ELISA) was used to determine the relative antigenicity of Neisseria meningitidis serogroup B antigens (B01smN, A05tmN, and NadA) in the MenPenta immunogenic composition.

[0730] Briefly, 96-well microtiter plates were coated with samples of immunogenic compositions obtained according to the heat stress protocol and diluted in carbonate-bicarbonate buffer at optimized starting antigen concentrations (except for NadA—see below). Samples were serially diluted onto the plates in eight-point serial dilutions and incubated overnight at 2°C–8°C. The following day, plates were washed three times with wash buffer (PBS 1× + 0.1% Tween-20) and then incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated specific monoclonal detection antibodies (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 Standards and Control (NIBSC)).

[0731] For NadA, a 96-well microtiter plate was coated with an in-house anti-NadA-specific monoclonal antibody diluted in carbonate-bicarbonate buffer and incubated overnight at 2°C–8°C. The next day, the plate was washed three 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 in eight-point serial dilutions onto the plate and incubated for 2 hours at room temperature. The plate was washed and then incubated with a second in-house NadA-specific HRP-conjugated monoclonal detection antibody for 1 hour at room temperature.

[0732] After a plate washing step, the plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate.

[0733] After 15 minutes (12 minutes for NadA), the reaction was stopped with 2N sulfuric acid (H2SO4) and the plates were read using a spectrophotometer. Color development was quantified by measuring the absorbance of each well at a wavelength of 450 nm with a reference wavelength of 540 nm. The magnitude of color development is directly proportional to the concentration of antigen obtained from the MenPenta immunogenic composition sample. The relative antigenicity (RA) of the sample was calculated by comparison with a reference standard lot using SoftMax Pro software. Relative antigenicity is the reportable value for this assay.

[0734] Measurement of free polysaccharide variation for serogroups A, C, W-135, and Y Free polysaccharides were measured by high performance anion exchange chromatography / pulsed amperometric detection (HPAEC-PAD) as indicated above.

[0735] Example 2: Settling onset time (T onset ) Settling start time (T onset ) is related to the flocculation properties of the suspension. onsetsuggests that above 60 minutes there is a high level of deflocculation, which may result in a denser cake and poorer cake resuspension properties of the formulation. onset It has been reported that AlPO4 formulations with a solubility time below 20 min are well agglomerated and exhibit better cake-forming properties (Muthurania, 2015).

[0736] 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, and pH 6.0 exhibited better suspension properties with longer settling onset times compared to formulations made with AlPO4 with a PZC greater than 5.

[0737] All of the formulations had a T of less than 20 minutes, which is indicative of a well-aggregated formulation. onset However, by reducing the PZC of the AlPO4 adjuvant, T onset Increasing T has several advantages, including better mixing and reconstitution of the adjuvant with the formulation, thus reducing the complexity of the filling process. onset A longer time supports the ease of handling of the formulation in the clinic, allowing it to maintain homogeneity over longer waiting times between mixing, drawing into a syringe and injection.

[0738] [Table 7]

[0739] Example 3: Pyrogenicity of dOMV formulated with mod-AlPO4 adjuvant or with AlPO4 adjuvant MenB was prepared with AlPO4PZC5.2 or mod-AlPO4PZC4.5 as disclosed above. The pyrogenicity of the compositions was determined as indicated above.

[0740] Increased temperature and pyrogenicity are known to be associated with dOMV components, and adsorption to aluminum adjuvants is known to reduce the pyrogenicity of dOMVs (Rosenqvist, 1998).

[0741] The dOMV adsorption rate was nearly 100% regardless of the PZC of AlPO (see Example 7). The adsorption rate of dOMV to mod-AlPO, with a PZC of approximately 4.5, showed similar IL-6 EC2 values compared to dOMV adsorbed to AlPO adjuvant, regardless of the dOMV dose used (high dose of 250 μg / mL or low dose of 50 μg / mL). 50 These results clearly demonstrate that the modification of the PZC by the AlPO4 adjuvant does not affect pyrogenicity.

[0742] [Table 8]

[0743] Example 4: Serum bactericidal activity of MenB immunogenic compositions hSBA response of fHBP On D0 and D42, bactericidal activity was measured in individual sera in IgG purified form, all collected from immunized rabbits, using human IgG / IgM depleted complement at a final concentration of 15%.

[0744] hSBA against the closely related fHBP A56 strain and the heterologous fHBP A22 strain As shown in Figures 1 and 3, in the absence of AlPO4 adjuvant, the MenB immunogenic composition A05tmN was able to induce both a moderate fHBP-specific hSBA response with a responder rate of 62.5 and a geometric mean titer (GMT) of 9 against the closely related variant fHBP A (A56; Figure 1) strain and a low fHBP-specific hSBA response with a 25% responder rate and a GMT of 4 against the heterologous variant fHBP A (A22; Figure 3) strain.

[0745] Furthermore, as shown in Figures 1 and 3, the presence of AlPO4 adjuvant in the MenB immunogenic composition increased the A05tmN-induced hSBA response by 5.1 (p-value = 0.001)-fold (GMT of 46) against the closely related A56 strain and 2.8 (p-value = 0.049)-fold (GMT of 12) against the heterologous A22 strain compared to the control (composition without adjuvant), whereas the mod-AlPO4 adjuvant further increased the response by 8.4 (p-value < 0.001)-fold (GMT of 76) against the closely related A56 strain and 4.9 (p-value = 0.004)-fold (GMT of 22) against the heterologous A22 strain.

[0746] Finally, AlPO4 adjuvant induced a % responder rate of 87.5% against the closely related A56 strain and 62.5% against the heterologous A22 strain, while mod-AlPO4 adjuvant induced a % responder rate of 100% against the closely related A56 strain and 87.5% against the heterologous A22 strain.

[0747] The results are summarized in Table 8 below.

[0748] [Table 9]

[0749] As can be seen in Figures 1 and 3, higher and more uniform hSBA responses (GMT and number of responders) to fHBP A were observed in formulations containing mod-AlPO4 adjuvant, but there was no statistical difference between mod-AlPO4 adjuvant and AlPO4 adjuvant.

[0750] hSBA against the closely related fHBP B44 strain and the heterologous fHBP B24 strain As shown in Figures 2 and 4, in the absence of AlPO4 adjuvant, the MenB immunogenic composition B01smN was able to induce a moderate fHBP-specific hSBA response with a 75% responder rate and a geometric mean titer (GMT) of 10 against the closely related variant fHBP B (B44; Figure 2) strain and a low fHBP-specific hSBA response with a 25% responder rate and a GMT of 4 against the heterologous variant fHBP B (B24; Figure 4) strain.

[0751] Furthermore, as shown in Figures 2 and 4, the presence of AlPO4 adjuvant in the MenB immunogenic composition increased the B01smN-induced hSBA response to the closely related B44 strain by 3.8 (p-value=0.011)-fold (GMT of 43) compared to the control (composition without adjuvant), but this did not induce an increase in the response to the heterologous B24 strain (GMT of 5). In contrast, the mod-AlPO4 adjuvant further increased the response to the closely related B44 strain by 6.2 (p-value=0.001)-fold (GMT of 64) and to the heterologous B24 strain by 3.2 (p-value=0.019)-fold (GMT of 14).

[0752] AlPO4 adjuvant induced a % responder rate of 100% against the closely related B44 strain and 25% against the heterologous B24 strain, while mod-AlPO4 adjuvant induced a % responder rate of 100% against the closely related B44 strain and 87.5% against the heterologous B24 strain.

[0753] The results are summarized in Table 9 below.

[0754] [Table 10]

[0755] As observed in Figures 2 and 4, a higher hSBA response was observed for fHBP B in formulations containing mod-AlPO4 adjuvant, which was statistically different from AlPO4 adjuvant for variant B24 (p-value = 0.043).

[0756] hSBA response to dOMV and NadA hSBA (dOMV response) against the homologous VR2-P1.2-PorA strain As illustrated in Figure 5, in the absence of adjuvant, dOMV was able to induce a high hSBA response against the homologous PorA VR2 P1.2 strain with a 100% responder rate and a GMT of 206.

[0757] The presence of AlPO4 or mod-AlPO4 adjuvant in the MenB immunogenic composition significantly increased the dOMV-induced hSBA response to the homologous VR2-P1.2-PorA strain, with a 4.9 (p-value = 0.007) and 4 (p-value = 0.015)-fold increase, GMTs of 875 and 775, and a % responder rate of 100%, respectively.

[0758] No statistical differences were observed between the AlPO4 adjuvant or mod-AlPO4 adjuvant responses.

[0759] hSBA against the same species of NadA strain As illustrated in Figure 6, in the absence of AlPO4 adjuvant, NadA was able to induce a high hSBA response with a GMT of 107 with a 100% responder rate against the homologous NadA1 strain.

[0760] The presence of AlPO4 or mod-AlPO4 adjuvant in MenB immunogenicity significantly increased the hSBA response to the homologous NadA strain due to NadA induction, with a 6.4 (p-value < 0.001) and 5.7 (p-value < 0.001) fold increase, GMTs of 698 and 652, and a % responder rate of 100%, respectively.

[0761] No statistical differences were observed between the AlPO4 adjuvant or mod-AlPO4 adjuvant responses.

[0762] conclusion The objective of this study was to compare the immunogenicity, based on the hSBA response, of a MenB immunogenic composition (A05tmN+B01smN+NadA+dOMV) formulated with or without unmodified AlPO4 adjuvant (PZC5.2) or phosphate-modified AlPO4 (PZC4.5) in New Zealand (NZ) White rabbits.

[0763] MenB immunogenic compositions formulated without AlPO4 were able to induce bactericidal activity with percent responder rates ranging from 25 to 100%. When formulated in the presence of either AlPO4 or mod-AlPO4 adjuvants, MenB immunogenic compositions were able to induce significantly higher hSBA titers compared to the groups without AlPO4 (all p-values ≤ 0.049), with a 2.8- to 8.4-fold increase depending on the strain used. For the B24 strain, a significantly higher response was observed in the presence of mod-AlPO4 adjuvant compared to the formulation containing AlPO4 (p-value = 0.019, fold increase = 3.2). Overall, higher and more uniform hSBA responses were observed in the presence of mod-AlPO4 adjuvant induced by A05tmN and B01smN fHBP. In terms of percent responder rates and geometric mean titers (GMTs), depending on the strain used, - 87.5-100% for MenB compositions with mod-AlPO4 adjuvant compared with 25-100% for MenB compositions with AlPO4 adjuvant. The GMT ranged from 14 to 76 with mod-AlPO4 adjuvant compared with 5 to 46 with AlPO4 adjuvant.

[0764] Example 5: Serum bactericidal activity of MenACWY immunogenic compositions with and without mod-AlPO4 adjuvant (PZC4.5) and MenPenta immunogenic compositions As shown in Figures 7-10, adsorption of the MenACWY immunogenic composition to mod-AlPO4 adjuvant (PZC4.5) did not adversely affect hSBA against the ACWY strain after the first and second doses.

[0765] For the MenACWY immunogenic composition, two doses are required to induce a strong hSBA response in rabbits. At D28 and D42, an increase in hSBA of the MenACWY immunogenic composition in the presence of mod-AlPO4 adjuvant (PZC4.5) was observed.

[0766] As shown in Figure 7, the presence of mod-AlPO4 adjuvant (PZC4.5) in the MenACWY immunogenic composition increased hSBA against strain A after the first and second doses. Compared to MenACWY alone, a significant increase in hSBA titers against strain A was observed in the presence of mod-AlPO4 adjuvant (21.2-fold at D28, p<0.001; 5.8-fold at D42, p<0.001). An even higher significant hSBA response was observed with the MenPenta (MenACWY+MenB) immunogenic composition formulated with AlPO4 adjuvant (15.4-fold at D28, p<0.001; 10.1-fold at D42, p<0.001).

[0767] No significant effect on hSBA titers against strain A was observed with the addition of MenB antigen (MenACWY+mod-AlPO4+MenB) compared with MenACWY+mod-AlPO4 adjuvant.

[0768] As shown in Figure 8, the presence of mod-AlPO4 adjuvant (PZC4.5) in the MenACWY immunogenic composition significantly increased hSBA titers against the C strain after the first and second doses. Compared to MenACWY alone, a significant increase in hSBA titers against the C strain was observed in the presence of mod-AlPO4 adjuvant (14.6-fold at D28, p<0.001; 3.6-fold at D42, p=0.008). An even higher significant hSBA response was observed with the MenPenta (MenACWY+MenB) immunogenic composition formulated with AlPO4 adjuvant (12.5-fold at D28, p<0.001; 3-fold at D42, p<0.022).

[0769] No significant effect on hSBA titers against strain C was observed with the addition of MenB antigen (MenACWY+mod-AlPO4+MenB) compared with MenACWY+mod-AlPO4 adjuvant.

[0770] As shown in Figure 9, the presence of mod-AlPO4 adjuvant (PZC4.5) in the MenACWY immunogenic composition increased hSBA titers against the W strain after the first and second doses. Compared to MenACWY alone, a significant increase in hSBA titers against the W strain was observed in the presence of mod-AlPO4 adjuvant (32.4-fold at D28, p<0.001; 8.1-fold at D42, p=0.001). An even higher significant hSBA response was observed with the MenPenta (MenACWY+MenB) immunogenic composition formulated with AlPO4 adjuvant (64.3-fold at D28, p<0.001; 3.1-fold at D42, p<0.022).

[0771] Compared with MenACWY+mod-AlPO4 adjuvant, the addition of MenB antigen (MenACWY+mod-AlPO4+MenB) did not significantly affect hSBA titers against the W strain.

[0772] As shown in Figure 10, the presence of mod-AlPO4 adjuvant (PZC4.5) in the MenACWY immunogenic composition increased hSBA against the Y strain after the first dose. Compared to MenACWY alone, a significant increase in hSBA titers against the Y strain was observed in the presence of mod-AlPO4 adjuvant (35.4-fold at D28, p<0.001; not significant at D42). An even higher significant hSBA response was observed with the MenPenta (MenACWY+MenB) immunogenic composition formulated with AlPO4 adjuvant (35-fold at D28, p<0.001; 4.4-fold at D42, p=0.01).

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

[0774] Example 6: Antigen stability in MenB and MenPenta immunogenic compositions MenB antigens (A05tm, B01sm, NadA and dOMV) The stability of individual antigens in MenB formulations was assessed by ELISA in AlPO4 or mod. AlPO4 at 37°C (NadA) and 45°C (BO1smN, A05tmN, and dOMV). Results indicated significantly greater stability of A05tmN when formulated in mod. AlPO4 compared with AlPO4 (p = 0.0003). NadA was also found to be significantly more stable in mod. AlPO4 compared with AlPO4 (p = 0.0082). While a slight improvement in stability was observed for BO1smN formulated in mod. AlPO4, no significant difference was observed between BO1smN formulated in mod. AlPO4 or AlPO4 (Figures 11A-C). No significant difference was observed between dOMV formulated in mod. AlPO4 or AlPO4 (results not shown).

[0775] This indicates that the MenB immunogenic composition has better antigenicity / potency over time under accelerated temperature conditions, thereby providing an improved shelf life.

[0776] Free polysaccharides of serogroups A, C, W, and Y in MenACWY or MenPenta immunogenic compositions As shown in Figures 12A-12D, no effect of AlPO4 adjuvant PZC modification was observed on the stability (monitored by free polysaccharide content) of serogroups A, C, W-135, and Y conjugates in MenACWY or MenPenta compositions compared to unadsorbed serogroups A, C, W, and Y (unadjuvanted MenACWY) at 45°C accelerated heat stress.

[0777] Unadsorbed A, C, W-135, and Y conjugates were stable for up to 4 years at 2-8°C, suggesting good long-term stability for A, C, W-135, and Y. The degradation profiles of unadsorbed A, C, W-135, and Y under accelerated heat stress in the presence of fHBP, NadA, and dOMV were similar to those adsorbed to modified PZC(4.5) AlPO4, suggesting good long-term stability of serogroups A, C, W-135, and Y in MenPenta immunogenic compositions (Figures 12A-D).

[0778] Example 7: Antigen adsorption onto AlPO Adsorption to AlPO4 by MenB antigen was assayed in compositions MenB or MenPenta at various PZCs (5.2 or 4.5-mod. AlPO4) and pH 6. The results are presented in Table 8.

[0779] [Table 11]

[0780] Example 8: Antigen stability in MenB immunogenic compositions The stability of antigens (A05tm, B01sm, NadA, and dOMV) in MenB immunogenic compositions formulated in 50 mM sodium acetate, 150 mM NaCl, pH 6.0 with mod-AlPO4 adjuvants with PZCs of 4.3, 4.5, and 4.8, and with AlPO4 adjuvant with PZC 5.2, was evaluated at 37°C (NadA) and 45°C (B01smN, A05tmN, and dOMV) for 30 days. Stability was determined by sandwich ELISA measurement of the antigenicity of MenB antigens according to the following protocol.

[0781] A 96-well microtiter plate was coated with an in-house anti-fHBP A05 monoclonal antibody, an in-house anti-fHBP B01 monoclonal antibody, an in-house anti-NadA monoclonal antibody, or an anti-porin B monoclonal antibody for dOMV (from NIBSC) and incubated overnight at 2°C–8°C. The next day, the plate was washed three times with wash buffer, followed by a blocking step. Following blocking, samples obtained according to the heat stress protocol were serially diluted onto the plate in eight-point serial dilutions and incubated for 2 hours at room temperature. The plate was washed and then incubated for 1 hour at room temperature with an HRP-conjugated detection monoclonal antibody consisting of a second in-house anti-fHBP A05 monoclonal antibody, a second in-house anti-fHBP B01 monoclonal antibody, a second in-house anti-NadA monoclonal antibody, or an anti-porin A monoclonal antibody for dOMV (from NIBSC).

[0782] Following a plate washing step, the plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate.

[0783] After the appropriate incubation period, the reaction was stopped with 2N sulfuric acid (H2SO4) and the plates were read using a spectrophotometer. Color development was quantified by measuring the absorbance of each well at a wavelength of 450 nm with a reference wavelength of 540 nm. The magnitude of color development is directly proportional to the concentration of antigen obtained from the MenB immunogenic composition sample.

[0784] Data were analyzed using SoftMax Pro GxP v6.5.1 software. An equivalent approach is to assess parallelism between the reference standard and the positive control, and between the reference standard and each test sample. The parallel line analysis (PLA) module available within the 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 reported value of "antigenic units per mL" (AU / mL) based on an arbitrary unit conversion relative to the reference standard.

[0785] As can be seen from Figures 13A-13D, the following was observed:

[0786] - A05tmN was significantly more stable in formulations containing AlPO4 with a PZC of ≤ 4.8 (ANCOVA: p<0.0001).

[0787] - NadA was significantly more stable in formulations containing AlPO4 with a PZC ≤ 4.5 (ANCOVA: p = 0.023).

[0788] - dOMV was significantly more stable in formulations containing AlPO4 with a PZC ≤ 4.5 (ANCOVA: p < 0.0169).

[0789] For B01sm, no significant differences were observed between the different formulations.

[0790] In conclusion, these results indicated that the stability of A05tmN was significantly increased 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. No significant differences were detected in the stability of B01smN formulated in AlPO4 with a PZC ranging from 4.3 to 5.2.

[0791] Taken together, these results indicate that MenB immunogenic compositions formulated with mod-AlPO4 adjuvants with a PZC below 5.0 exhibit greater antigenicity / potency stability over time under accelerated temperature conditions, thereby providing improved shelf life.

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Caron F,du Chatelet IP,Leroy JP,Ruckly C,Blanchard M,Bohic N,et al.From tailor-made to ready-to-wear meningococcal B vaccines:longitudinal study of a clonal meningococcal B outbreak.Lancet Infect Dis.2011;11(6):455-63. Christensen H,May M,Bowen L,Hickman M,Trotter CL.Meningococcal carriage by age:a systematic review and meta-analysis.Lancet Infect Dis.2010;10(12):853-61. Diminsky,D.M.(1999).Physical,chemical and immunological stability of CHO-derived hepatitis B surface antigen(HBsAg)particles.Vaccine,18(1-2),3-17.doi:10.1016 / s0264-410x(99)00149-8 Dyet KH,Martin DR.Clonal analysis of the serogroup B meningococci causing New Zealand’s epidemic.Epidemiol Infect.2006;134(2):377-83. Folaranmi T,Rubin L,Martin SW,Patel M,MacNeil JR,Centers for Disease C.Use of Serogroup B Meningococcal Vaccines in Persons Aged> / =10 Years at Increased Risk for Serogroup B Meningococcal Disease:Recommendations of the Advisory Committee on Immunization Practices,2015.MMWR Morb Mortal Wkly Rep.2015;64(22):608-12. Fredriksen JH,Rosenqvist E,Wedege E,Bryn K,Bjune G,Froholm LO,et al.Production,characterization and control of MenB-vaccine “Folkehelsa”:an outer membrane vesicle vaccine against group B meningococcal disease.NIPH Ann.1991;14(2):67-79;discussion -80. Germinario C,Tafuri S,Napoli C,Montagna MT,Balducci MT,Fortunato F,et al.Young-adult carriers of Neisseria meningitidis in Puglia(Italy):will the pattern of circulating meningococci change following the introduction of meningococcal serogroup C conjugate vaccines? Hum Vaccin.2010;6(12):1025-7. Grodet C,Dequin PF,Watt S,Lanotte P,de Gialluly C,Taha MK,et al.Outbreak in France of Neisseria meningitidis B:15:P1.12 belonging to sequence type 1403.Clin Microbiol Infect.2004;10(9):845-8. Harrison L,Granoff D,Pollard A.Meningococcal capsular group A,C,W,and Y conjugate vaccines.[ed.]Orenstein WA,Offit PA,Edwards KM Plotkin SA.Vaccines.7.Philadelphia(PA):Elsevier;2018.p.619-43. Harrison OB,Claus H,Jiang Y,Bennett JS,Bratcher HB,Jolley KA,et al.Description and nomenclature of Neisseria meningitidis capsule locus.Emerg Infect Dis.2013;19(4):566-73. Hem,S.L.(2007).Imject Alum is not aluminum hydroxide adjuvant or aluminum phosphate adjuvant.Vaccine,25(27),4985-4986.doi:10.1016 / j.vaccine.2007.04.078 Hem,S.L.(2007).Relationship between physical and chemical properties of aluminum-containing adjuvants and immunopotentiation.Expert Rev Vaccines,6(5),685-698.doi:10.1586 / 14760584.6.5.685 Iyer,S.R.(2004).Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant.Vaccine,22(11-12),1475-1479.doi:10.1016 / j.vaccine.2003.10.023 Jones,L.S.(2005).Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens.The Journal of biological chemistry,280(14),13406-13414.doi:10.1074 / jbc.M500687200 Kvalsvig AJ,Unsworth DJ.The immunopathogenesis of meningococcal disease.J Clin Pathol.2003;56(6):417-22. Maa,Y.F.(2003).Stabilization of alum-adjuvanted vaccine dry powder formulations:mechanism and application.Journal of pharmaceutical sciences,92(2),319-332.doi:10.1002 / jps.10294 MacLennan J,Kafatos G,Neal K,Andrews N,Cameron JC,Roberts R,et al.Social behavior and meningococcal carriage in British teenagers.Emerg Infect Dis.2006;12(6):950-7. Maiden MC,Ibarz-Pavon AB,Urwin R,Gray SJ,Andrews NJ,Clarke SC,et al.Impact of meningococcal serogroup C conjugate vaccines on carriage and herd immunity.J Infect Dis.2008;197(5):737-43. Muthurania,K.I.(2015).Investigation of the Sedimentation Behavior of Aluminum Phosphate:Influence of pH,Ionic Strength,and Model Antigens.Journal of pharmaceutical sciences,104(11),3770-3781.doi:10.1002 / jps.24584 Pace D,Pollard AJ.Meningococcal disease:clinical presentation and sequelae.Vaccine.2012;30 Suppl 2:B3-9. Rinella,J.V.(1996).Treatment of aluminium hydroxide adjuvant to optimize the adsorption of basic proteins.Vaccine,14(4),298-300.doi:10.1016 / 0264-410x(95)00194-6 Rodriguez AP,Dickinson F,Baly A,Martinez R.The epidemiological impact of antimeningococcal B vaccination in Cuba.Mem Inst Oswaldo Cruz.1999;94(4):433-40. Rosenqvist,E.H.(1998).Effect of aluminium hydroxide and meningococcal serogroup C capsular polysaccharide on the immunogenicity and reactogenicity of a group B Neisseria meningitidis outer membrane vesicle vaccine.Developments in biological standardization,92,323-333. Rouphael NG,Stephens DS.Neisseria meningitidis:biology,microbiology,and epidemiology.Methods Mol Biol.2012;799:1-20. Seeber,S.J.(1991).Predicting the adsorption of proteins by aluminium-containing adjuvants.Vaccine,9(3),201-203.doi:10.1016 / 0264-410x(91)90154-x Stephens DS,Apicella MA.Neisseria meningitidis.[ed.]J.E.Bennett,R.Dolin and M.J.Blaser.Philadelphia:Elsevier Saunders;2015.p.2425-45. 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Claims

1. A combination of Neisseria meningitidis serogroup B antigens, comprising at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and aluminum hydroxyphosphate (AlPO ４ ) adjuvant, aluminum hydroxyphosphate (AlPO) selected as having a point of zero charge (PZC) below 5 ４ ) an adjuvant.

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

5.

3. AlPO ４ The immunogenic composition of claim 1 or 2, wherein the difference between the PZC of the 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, having a pH in the range of about 5.5 to about 7.0, or about 6.

0.

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

6. The fHBP may be AlPO in an amount of 85% or less of the total amount of fHBP in the composition, or in an amount ranging from about 50% to less than 85% of the total amount of fHBP in the composition. ４ The immunogenic composition according to any one of claims 1 to 5, which is adsorbed onto

7. 7. The immunogenic composition of claim 1, wherein the fHBP B has an isoelectric point (pI) ranging 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 of any one of claims 1 to 7, wherein the fHBP B is non-lipidated.

9. The immunogenic composition of any one of claims 1 to 8, wherein the fHBP B is a mutant fHBP B comprising at least one mutation that reduces or inhibits 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 mutant fHBP B comprising at least about 85% identity to SEQ ID NO:

3.

11. 11. The immunogenic composition of any one of claims 1 to 10, wherein the fHBP B is a mutant fHBP B comprising at least one amino acid substitution selected from at least one of: a) an amino acid substitution of glutamine (Q38) at amino acid 38; b) an amino acid substitution of glutamic acid (E92) at amino acid 92; c) an amino acid substitution of arginine (R130) at amino acid 130; d) an amino acid substitution of serine (S223) at amino acid 223; and e) an amino acid substitution of histidine (H248) at amino acid 248, based on the numbering of SEQ ID NO: 6, or comprises or consists of SEQ ID NO: 4, or comprises or consists of SEQ ID NO:

9.

12. 12. The immunogenic composition of any one of claims 1 to 11, wherein the fHBP A has an isoelectric point (pI) ranging 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 of any one of claims 1 to 12, wherein the fHBP A is non-lipidated.

14. The immunogenic composition of any one of claims 1 to 13, wherein the fHBP A is a mutant fHBP A comprising at least one mutation that reduces or inhibits 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. 16. The immunogenic composition of any one of claims 1 to 15, wherein the fHBP A is a mutant fHBP A comprising at least one amino acid substitution selected from at least one 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 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 or consisting of SEQ ID NO: 2, or comprising or consisting of SEQ ID NO:

8.

17. 17. The immunogenic composition of any one of claims 1 to 16, wherein the fHBP A and / or the fHBP B are 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, respectively.

18. 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 with SEQ ID NO: 5 or comprises or consists of SEQ ID NO:

5.

19. 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 at about 50 μg / dose.

20. The immunogenic composition of any one of claims 5 to 19, wherein the dOMV comprises Porin A (PorA) protein.

21. 21. The immunogenic composition of claim 20, wherein the Porin A (PorA) protein is selected from the PorA VR2 subtype or is PorA VR2 P1.

2.

22. 22. The immunogenic composition of any one of claims 5 to 21, wherein the dOMVs are 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 at 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, further comprising a buffer.

24. 24. The immunogenic composition of claim 23, wherein the buffer is selected from among Tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer, or histidine buffer.

25. 25. The immunogenic composition of claim 23 or 24, wherein the buffer is a sodium acetate buffer.

26. 25-100 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, 25-100 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, 25-100 μg / dose of NadA protein consisting of SEQ ID NO: 5, 20-250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, 100-800 μg / dose of AlPO selected as having a PZC of about 4.

5. ４ 26. The immunogenic composition of any one of claims 1 to 25, comprising or consisting of an adjuvant, 50 mM acetate buffer, pH 6.

0.

27. 27. The immunogenic composition of any one of claims 1 to 26, further comprising at least capsular saccharides from one or more of Neisseria meningitidis serogroups A, C, W135 and / or Y conjugated to a carrier protein.

28. 28. The immunogenic composition of claim 27, wherein the conjugated capsular saccharide is conjugated to a tetanus toxoid carrier.

29. Settling onset time (T) ranges from about 3.5 minutes to about 10 minutes. ｏｎｓｅｔ The immunogenic composition of any one of claims 1 to 28, comprising:

30. 30. The immunogenic composition of any one of claims 1 to 29, which enhances an immune response against N. meningitidis serogroup B strains that express a fHBP B heterologous to the fHBP B of the composition.

31. The immunogenic composition of any one of claims 6 to 30, which enhances the stabilization of at least one of fHBP A and NadA proteins.

32. A vaccine comprising the immunogenic composition of any one of claims 1 to 31.

33. An immunogenic composition according to any one of claims 1 to 31 or a vaccine according to claim 32 for use in a method of inducing an immune response against a group B strain of Neisseria meningitidis.

34. 1. An AlPO having a PZC of less than 5 for enhancing the immune response induced by a composition comprising a N. meningitidis fHBP B antigen against a N. meningitidis serogroup B strain expressing a fHBP B antigen heterologous to the fHBP B antigen of the composition. ４ Use of adjuvants.

35. AlPO having a PZC below 5 for stabilizing at least one of fHBP A and NadA proteins in an immunogenic composition ４ Use of adjuvants.

36. The time to onset of sedimentation (T) of a 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 is ｏｎｓｅｔ ) in a range of about 3.5 minutes to about 10 minutes to stabilize the AlPO ４ Use of adjuvants.

37. AlPO 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. ４ Use of adjuvants.

38. 1. A method for producing 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, the method comprising the steps of: ４ Use of adjuvants.

39. AlPO ４ The use according to any one of claims 34 to 38, wherein the adjuvant is one according to claim 2 or 3.

40. 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 combination of Neisseria meningitidis serogroup B antigens, comprising at least one factor H binding protein (fHBP) A and one factor H binding protein (fHBP) B, and AlPO ４ and an adjuvant, comprising at least: a) AlPO having a PZC below 5 ４ selecting an adjuvant; b) the AlPO selected in step a) ４ combining an adjuvant with at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, said combining being carried out in any order; A method comprising:

42. A method for stabilizing at least one of fHBP A and NadA proteins in an immunogenic composition, comprising at least a) AlPO having a PZC below 5 ４ selecting an adjuvant; b) the AlPO selected in step a) ４ combining an adjuvant with the fHBP A or NadA protein; c) obtaining an immunogenic composition in which the fHBP A or NadA protein is stabilized; A method comprising:

43. 1. A method for preparing an immunogenic composition comprising an N. meningitidis fHBP B antigen, the method inducing an enhanced immune response against a N. meningitidis serogroup B strain expressing a fHBP B heterologous to the fHBP B antigen of the composition, the method comprising at least: a) AlPO having a PZC below 5 ４ selecting an adjuvant; b) the AlPO selected in step a) ４ combining an adjuvant with the fHBP B antigen; c) obtaining said immunogenic composition; A method comprising:

44. AlPO ４ The method according to any one of claims 41 to 43, wherein the adjuvant is that according to claim 2 or 3.

45. The method of any one of claims 42 to 45, wherein the immunogenic composition is according to any one of claims 1 to 31.

46. 33. A method of inducing an immune response against a Neisseria meningitidis serogroup B strain in an individual in need thereof, comprising administering to the individual at least the immunogenic composition of any one of claims 1 to 31 or the vaccine of claim 32, wherein said administering induces an immune response against the Neisseria meningitidis serogroup B strain.

33. A method of enhancing an immune response induced by a composition comprising a N. meningitidis fHBP B antigen against a N. meningitidis serogroup B strain expressing a fHBP B heterologous to the fHBP B antigen of the composition in an individual in need thereof, the method comprising administering to the individual at least an immunogenic composition of any one of claims 1 to 31 or a vaccine of claim 32, wherein the administering step induces an enhanced immune response against the N. meningitidis serogroup B strain expressing the heterologous fHBP B.

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