Meningococcal compositions and methods thereof

Abstract

To provide a cross-protective vaccine or composition effective against various MnB and meningococcal serotypes A, C, Y, and W, and / or X isolates.SOLUTION: In one aspect, the present disclosure relates to compositions comprising a factor H binding protein (fHBP) and a meningococcal non-serotype B capsular polysaccharide, and methods of use thereof. The disclosure further relates to the use of compositions comprising fHBP, e.g. for eliciting an immune response against meningococcal serogroup B strains and non-serogroup B strains. The compositions and methods described herein are intended for administration in humans, including adults, adolescents, young children and infants.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 907,097, filed September 27, 2019, and U.S. Provisional Patent Application No. 63 / 040,498, filed June 17, 2020. All of the foregoing applications are incorporated by reference herein in their entirety.

[0002] The present disclosure relates to Neisseria meningitidis compositions and methods thereof. [Background technology]

[0003] Neisseria meningitidis is a Gram-negative, encapsulated bacterium that can cause septicemia, meningitis, and death. Neisseria meningitidis can be classified into at least 12 serotypes (including serotypes A, B, C, 29E, H, I, K, L, W-135 (now mostly called W), X, Y, and Z) based on chemically and antigenically distinct capsular polysaccharides. Strains with five of the serotypes (A, B, C, Y, and W135) cause the majority of disease.

[0004] Meningococcal meningitis is a devastating disease that can kill children and young adults within hours despite the availability of antibiotics. There is a need for improved immunogenic compositions against meningococcal serotypes A, B, C, Y, and W135 and / or X. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2012 / 032489 [Patent Document 2] U.S. Patent Publication No. 20120093852 [Patent Document 3] International Publication No. WO2013 / 132452 [Patent Document 4] U.S. Patent Publication No. 20160030543 [Patent Document 5] WO2012032489 No. [Patent Document 6] U.S. Patent Publication No. 2012 / 0093852 [Patent Document 7] U.S. Patent No. 10,183,070 [Patent Document 8] WO04 / 083251 issue [Patent Document 9] No. US4709017 [Patent Document 10] No. US4950740 [Patent Document 11] No. US5917017 [Patent Document 12] No. US6455673 [Patent Document 13] No. US5843711 [Patent Document 14] EP0372501 issue [Patent Document 15] EP0378881 issue [Patent Document 16] EP0427347 issue [Patent Document 17] WO93 / 17712 [Patent Document 18] WO94 / 03208 [Patent Document 19] WO98 / 58668 issue [Patent Document 20] EP0471177 issue [Patent Document 21] WO91 / 01146 issue [Patent Document 22] WO02 / 091998 issue [Patent Document 23] WO01 / 72337 issue [Patent Document 24] WO00 / 61761 issue [Patent Document 25] EP594610 issue

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Non-licensed literature

[0006] [Non-licensed document 1] Uchida, J. Biol. Chem., Volume 218; Pages 3838~3844, 1973 [Non-patent document 2] Nicholls and Youle, Genetically Engineered Toxins, edited by Frankel, Maecel Dekker Inc, 1992 [Non-patent document 3] Kuo et al. (1995) Infect Immun, 63:2706-13 [Non-patent document 4] Baraldoi et al. (2004) Infect Immun, 72:4884-7 [Non-Patent Document 5] Falugi et al. (2001) Eur J Immunol 31:3816-3824 [Non-patent document 6] Lowry et al. (1951) J. Biol. Chem., 193, 265-275 [Non-Patent Document 7] Peterson et al., Analytical Biochemistry, 100, 201-220 (1979) [Non-patent document 8] Monsigny et al. (1988) Anal. Biochem., 175, 525-530 [Non-Patent Document 9] Gever et al. (1979) Med. Microbiol. Immunol., 165:171-288 [Non-Patent Document 10] Bethell et al., J. Biol. Chem., 1979, 254;2572-4 [Non-Patent Document 11] Hearn et al., J. Chromatogr., 1981, 218:509-18 [Non-Patent Document 12] Chu C. et al., Infect.Immunity, 1983, pp. 245-256 [Non-Patent Document 13] Egan et al., Vaccine, 27(24):3175-3180 (2009) Summary of the Invention [Problem to be solved by the invention]

[0007] Currently, there are no commercially available cross-protective vaccines or compositions that are effective against a wide range of MnB and meningococcal serogroups A, C, Y, and W, and / or X isolates. Thus, there is a need for cross-protective vaccines or compositions that are effective against a wide range of MnB and meningococcal serogroups A, C, Y, and W, and / or X isolates.

[0008] A further object of the present disclosure is to provide improved schedules for administering meningococcal vaccines. Under the current recommended scheme, there are 4-5 vaccinations given against meningococcal serotypes A, C, W, Y, and B, given at various ages. There is an unmet need for efficient vaccinations that could simplify immunization schedules and improve vaccination rates to achieve further reductions in invasive meningococcal disease (IMD). [Means for solving the problem]

[0009] To meet these and other needs, the present disclosure relates to meningococcal compositions and methods. The inventors have now discovered a method of inducing an immune response in a human, the method comprising the step of administering to the human a composition comprising: a) a polypeptide derived from meningococcal factor H binding protein (fHBP); (b) a meningococcal serogroup A capsular saccharide conjugate; (c) a meningococcal serogroup C capsular saccharide conjugate; (d) a meningococcal serogroup W capsular saccharide conjugate; and (e) a meningococcal serogroup Y capsular saccharide conjugate, wherein the composition induces an immune response against at least one of meningococcal serogroups A, C, W-135 and Y capsular polysaccharides, and meningococcal serogroup B, wherein the immune response comprises a titer of serum bactericidal antibodies that is higher than the titer of serum bactericidal antibodies induced by the respective licensed vaccines against the serogroups.

[0010] The inventors have surprisingly found a method of inducing an immune response in a human, the method comprising the step of administering to the human a composition comprising: (a) a first polypeptide derived from meningococcal factor H binding protein (fHBP); (b) a second polypeptide derived from meningococcal factor H binding protein (fHBP); (c) a meningococcal serogroup A capsular saccharide conjugate; (d) a meningococcal serogroup C capsular saccharide conjugate; (e) a meningococcal serogroup W capsular saccharide conjugate; and (f) a meningococcal serogroup Y capsular saccharide conjugate, wherein the composition induces an immune response against at least one of meningococcal serogroups A, C, W-135 and Y capsular polysaccharides and meningococcal serogroup B, wherein the immune response comprises a titer of serum bactericidal antibodies that is higher than the titer of serum bactericidal antibodies induced by the respective licensed vaccines against the serogroups.

[0011] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity to any one amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:62. In a preferred aspect, the composition comprises: (a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2; (c) a meningococcal serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; (d) a meningococcal serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (e) a meningococcal serogroup C capsular saccharide conjugated to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker. and (f) a meningococcal serogroup Y capsular saccharide directly conjugated to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, in the absence of a linker.

[0012] In some embodiments, the composition elicits an immune response against any one of meningococcal serotypes A, C, W-135, and Y, wherein the serum bactericidal antibody response is greater than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines.

[0013] In some embodiments, the composition elicits an immune response against meningococcal serotype A, wherein the serum bactericidal antibody response is higher than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines.

[0014] In some embodiments, the composition elicits an immune response against meningococcal serotype C, wherein the serum bactericidal antibody response is higher than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines.

[0015] In some embodiments, the composition elicits an immune response against meningococcal serotype W, wherein the serum bactericidal antibody response is higher than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines.

[0016] In some embodiments, the composition elicits an immune response against meningococcal serotype Y, wherein the serum bactericidal antibody response is higher than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines.

[0017] In some embodiments, the composition elicits an immune response against each of Neisseria meningitidis serotypes A, C, W-135, and Y, wherein the serum bactericidal antibody response is greater than that elicited by licensed Neisseria meningitidis serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines.

[0018] In some embodiments, the composition elicits an immune response against meningococcal serogroup B, wherein the serum bactericidal antibody response is higher than that elicited by a licensed meningococcal serogroup B H factor conjugate vaccine.

[0019] In some embodiments, the composition elicits an immune response against each of meningococcal serotypes A, C, W-135, and Y, wherein the serum bactericidal antibody response against each of meningococcal serotypes A, C, W-135, and Y capsular polysaccharides is greater than that elicited by a licensed meningococcal serotype A, C, W-135, and Y meningococcal capsular polysaccharide vaccine; and the composition elicits an immune response against meningococcal serotype B, wherein the serum bactericidal antibody response is greater than that elicited by a licensed meningococcal serotype B H factor conjugate vaccine, wherein the licensed meningococcal serotype A, C, W-135, and Y meningococcal capsular polysaccharide vaccine and the licensed meningococcal serotype B H factor conjugate vaccine are administered sequentially rather than as a combined dose.

[0020] In some embodiments, the composition includes an adjuvant. In some embodiments, the composition includes an aluminum adjuvant. In some embodiments, the composition includes aluminum hydroxide. In some embodiments, the composition includes aluminum phosphate. In some embodiments, the composition includes aluminum.

[0021] In some embodiments, at least 90% of the first polypeptide is aluminum bound in the composition, hi some embodiments, at least 90% of the second polypeptide is aluminum bound in the composition.

[0022] In some embodiments, the composition is formulated as a sterile liquid. In some embodiments, the composition includes a pharmaceutically acceptable preservative. In some embodiments, the composition includes polysorbate-80. In some embodiments, the composition includes Tris-HCl, sodium chloride, sucrose, histidine, polysorbate 80, and aluminum phosphate.

[0023] In some embodiments, the composition comprises about 120 μg / ml of a first polypeptide, about 120 μg / ml of a second polypeptide, about 0.5 mg / ml aluminum as aluminum phosphate, about 0.02 mg polysorbate-80, about 10 mM histidine, and about 150 mM sodium chloride.

[0024] In some aspects, the composition comprises about 60 μg of the first polypeptide, about 60 μg of the second polypeptide, about 5 μg of MenA capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenC capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenW capsular saccharide conjugated to about 3.75 μg of TT, about 5 μg of MenY capsular saccharide conjugated to about 3.25 μg of TT, about 97 μg of Tris-HCl, pH 6.8±0.3, 4.69-4.71 mg of sodium chloride, about 28 mg of sucrose, about 0.78 mg of L-histidine, about 0.02 mg of polysorbate-80, about 0.25 mg of aluminium, and further comprising 0.5 mL of water per dose.

[0025] In some embodiments, the immune response comprises serum bactericidal antibodies. In some embodiments, the composition is capable of eliciting a booster immune response against at least one of meningococcal serotypes A, C, W-135, and Y. In some embodiments, the composition is capable of eliciting a booster immune response against meningococcal serotype B.

[0026] In some embodiments, the immune response is elicited in humans up to 25 years of age. In some embodiments, the immune response is elicited in humans at least 2 months to 25 years of age. In some embodiments, the immune response is elicited in humans aged 10 to 25 years. In some embodiments, the immune response is elicited in humans aged 10 to 26 years. In some embodiments, the immune response is elicited in humans aged 12 months to under 18 months or 18 months to under 24 months. In some embodiments, the immune response is elicited in humans aged 18 months to under 24 months. In some embodiments, the immune response is elicited in humans aged 24 months or older but under 10 years.

[0027] In some embodiments, the immune response is elicited in humans who are seronegative for meningococcal serogroups A, C, W-135, and Y. In some embodiments, the immune response is elicited in humans who are seropositive for meningococcal serogroups A, C, W-135, and Y.

[0028] In some embodiments, the composition is administered to the human in at least two doses, the second dose being about 6 months after the first dose. In some embodiments, the human is at least 10 years old and at most 17 years old. In some embodiments, a third dose of the composition is administered to the human, and the human is at least 16 years old.

[0029] In some embodiments, the composition is administered to a human in up to two doses, the second dose being about six months after the first dose. In some embodiments, the composition induces an immune response against A22. In some embodiments, the composition induces an immune response against A56. In some embodiments, the composition induces an immune response against B24. In some embodiments, the composition induces an immune response against B44.

[0030] In some aspects, the composition comprises about 60 μg of the first polypeptide, about 60 μg of the second polypeptide, about 5 μg of MenA capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenC capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenW capsular saccharide conjugated to about 3.75 μg of TT, about 5 μg of MenY capsular saccharide conjugated to about 3.25 μg of TT, about 97 μg of Tris-HCl, pH 6.8±0.3, 4.69-4.71 mg of sodium chloride, about 28 mg of sucrose, about 0.78 mg of L-histidine, about 0.02 mg of polysorbate-80, about 0.25 mg of aluminium, and further comprising 0.5 mL of water per dose.

[0031] 1. A composition comprising: (a) a first polypeptide derived from a meningococcal factor H binding protein (fHBP); (b) a second polypeptide derived from a meningococcal factor H binding protein (fHBP); (c) a meningococcal serogroup A capsular saccharide conjugate; (d) a meningococcal serogroup C capsular saccharide conjugate; (e) a meningococcal serogroup W capsular saccharide conjugate; and (f) a meningococcal serogroup Y capsular saccharide conjugate, wherein the composition elicits an immune response against at least one of meningococcal serogroups A, C, W-135, and Y, and wherein the serum bactericidal antibody response is greater than that elicited by a licensed vaccine against the meningococcal serogroups.

[0032] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity to any one amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:62. (a) a first polypeptide derived from a meningococcal factor H binding protein (fHBP); (b) a second polypeptide derived from a meningococcal factor H binding protein (fHBP); (c) a meningococcal serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (d) a meningococcal serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry. 1. A composition comprising (a) a meningococcal serogroup C capsular saccharide that is conjugated to a tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker; (b) a meningococcal serogroup W capsular saccharide that is conjugated to a tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker; and (c) a meningococcal serogroup Y capsular saccharide that is conjugated to a tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker, wherein the composition elicits an immune response against at least one of meningococcal serogroups A, C, W-135 and Y, and wherein the serum bactericidal antibody response is greater than that elicited by a licensed meningococcal serogroup A, C, W-135 and Y meningococcal capsular polysaccharide vaccine.

[0033] A method of inducing an immune response in a human against Neisseria meningitidis serogroup B subfamily A strains and Neisseria meningitidis serogroup B subfamily B strains, comprising administering to the human an effective amount of the composition.

[0034] A method of inducing an immune response in a human against meningococcal serogroup A, meningococcal serogroup C, meningococcal serogroup W, and / or meningococcal serogroup Y strains, comprising administering to the human an effective amount of the composition.

[0035] A method for inducing an immune response in a human against meningococcal serogroup A, meningococcal serogroup B, meningococcal serogroup C, meningococcal serogroup W, and / or meningococcal serogroup Y strains, comprising administering to the human an effective amount of a composition.

[0036] A method for inducing an immune response in a human against Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W, Neisseria meningitidis serogroup Y strains, and / or Neisseria meningitidis serogroup X strains, comprising the step of administering an effective amount of the composition to the human.

[0037] In some aspects, the patient has not previously received a multivalent meningococcal capsular saccharide-carrier protein conjugate vaccine prior to the first administration of the composition. In some aspects, the patient has previously received a multivalent meningococcal capsular saccharide-carrier protein conjugate vaccine prior to the first administration of the composition. [Brief explanation of the drawings]

[0038] [Figure 1A]Figure 1 shows immune responses to MenB test strains one month after dose 2, as measured in hSBA. Error bars represent 95% Cis. MenB strains are noted with FHbp variants in parentheses. *MenABCWY, n=418-4432; MenB-FHbp, n=814-850. + Combined response for all four MenB test strains = hSBA titer ≥ LLOQ. ++MenABCWY, n=227-262; MenACWY-CRM, n=446-506. §MenABCWY, n=187-257; MenACWY-CRM, n=370-495. hSBA = serum bactericidal activity using human complement; MenA = meningococcal serotype A; LLOQ = lower limit of quantitation; m = months; MenABCWY = pentavalent serotypes A, B, C, W, and Y vaccine; MenACWY-CRM = MENVEO®, meningococcal (serotypes A, C, Y, and W-135) oligosaccharide CRM197 conjugate vaccine (tetravalent meningococcal CRM vaccine); MenB = meningococcal serotype B; MenB-FHbp = TRUMENBA®, meningococcal B bivalent recombinant lipoprotein 2086 (bivalent rLP2086) vaccine; MenC = meningococcal serotype C; MenW = meningococcal serotype W; MenY = meningococcal serotype Y; PD = post-dose. [Figure 1B]Figure 1 shows immune responses to MenA, MenC, MenW, and MenY test strains 1 month after doses 1 and 2, as measured in hSBA. Error bars represent 95% Cis. MenB strains note FHbp variants in parentheses. *MenABCWY, n=418-4432; MenB-FHbp, n=814-850. + Combined response for all four MenB test strains = hSBA titer ≥ LLOQ. ++MenABCWY, n=227-262; MenACWY-CRM, n=446-506. §MenABCWY, n=187-257; MenACWY-CRM, n=370-495. hSBA = serum bactericidal activity using human complement; MenA = meningococcal serotype A; LLOQ = lower limit of quantitation; m = months; MenABCWY = pentavalent serotypes A, B, C, W, and Y vaccine; MenACWY-CRM = MENVEO®, meningococcal (serotypes A, C, Y, and W-135) oligosaccharide CRM197 conjugate vaccine (tetravalent meningococcal CRM vaccine); MenB = meningococcal serotype B; MenB-FHbp = TRUMENBA®, meningococcal B bivalent recombinant lipoprotein 2086 (bivalent rLP2086) vaccine; MenC = meningococcal serotype C; MenW = meningococcal serotype W; MenY = meningococcal serotype Y; PD = post-dose. [Figure 2A] Figure 1 shows local reactions reported within 7 days after any dose. MenABCWY, n=542; MenB-FHbp+MenACWY-CRM, n=1050. *At MenABCWY / MenB-FHbp injection site. MenABCWY = pentavalent serotypes A, B, C, W, Y vaccine; MenACWY-CRM = MENVEO®, meningococcal (serotypes A, C, Y, and W-135) oligosaccharide CRM197 conjugate vaccine (tetravalent meningococcal CRM vaccine); MenB-FHbp = TRUMENBA®, meningococcal B bivalent recombinant lipoprotein 2086 (bivalent rLP2086). [Figure 2B]Figure 1 shows systemic events reported within 7 days after any dose. MenABCWY, n=542; MenB-FHbp+MenACWY-CRM, n=1050. *At MenABCWY / MenB-FHbp injection site. MenABCWY = pentavalent serotypes A, B, C, W, Y vaccine; MenACWY-CRM = MENVEO®, meningococcal (serotypes A, C, Y, and W-135) oligosaccharide CRM197 conjugate vaccine (tetravalent meningococcal CRM vaccine); MenB-FHbp = TRUMENBA®, meningococcal B bivalent recombinant lipoprotein 2086 (bivalent rLP2086). [Figure 3] Estimated number of cases averted over 10 years under various vaccine administration strategies compared with a hypothetical no-vaccination scenario.

[0039] Sequence Identifier SEQ ID NO: 1 shows the amino acid sequence of the recombinant Neisseria meningitidis, serotype B, 2086 variant A05 polypeptide antigen. SEQ ID NO: 2 shows the amino acid sequence of the recombinant Neisseria meningitidis, serotype B, 2086 variant B01 polypeptide antigen. SEQ ID NO:3 shows the amino acid residues at positions 1 to 4 of SEQ ID NO:1 and SEQ ID NO:2. SEQ ID NO: 4 shows the amino acid sequence of the N-terminus of the recombinant Neisseria subfamily A LP2086 polypeptide (rLP2086) (A05) polypeptide. SEQ ID NO: 5 shows the amino acid sequence of the N-terminus of the Neisseria subfamily A LP2086 M98250771 polypeptide (A05) polypeptide. SEQ ID NO: 6 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B153. SEQ ID NO: 7 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A04. SEQ ID NO: 8 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A05. SEQ ID NO: 9 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A12. SEQ ID NO: 10 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A22. SEQ ID NO: 11 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B02. SEQ ID NO: 12 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B03. SEQ ID NO: 13 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B09. SEQ ID NO: 14 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B22. SEQ ID NO: 15 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B24. SEQ ID NO: 16 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B44. SEQ ID NO: 17 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B16. SEQ ID NO: 18 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A07. SEQ ID NO: 19 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A19. SEQ ID NO: 20 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A06. SEQ ID NO: 21 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A15. SEQ ID NO: 22 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A29. SEQ ID NO: 23 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B15. SEQ ID NO: 24 shows the amino acid sequence of the N-terminus of the recombinant Neisseria subfamily B LP2086 polypeptide (rLP2086) (B01) polypeptide. SEQ ID NO: 25 shows the amino acid sequence of the N-terminus of the Neisseria subfamily B LP2086 CDC-1573 polypeptide (B01) polypeptide. SEQ ID NO: 26 shows the amino acid sequence of a meningococcal serogroup A strain expressing factor H binding protein (fHBP) B16. SEQ ID NO: 27 shows the amino acid sequence of a meningococcal serogroup C strain expressing fHBP A10. SEQ ID NO: 27 also shows the amino acid sequence of a meningococcal serogroup W strain expressing fHBP A10. SEQ ID NO: 28 shows the amino acid sequence of a meningococcal serogroup W strain expressing fHBP A19. SEQ ID NO: 29 shows the amino acid sequence of a meningococcal serotype Y strain expressing fHBP B47. SEQ ID NO: 30 shows the amino acid sequence of a meningococcal serotype X strain expressing fHBP B49 . SEQ ID NO: 31 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B16. SEQ ID NO: 32 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A07. SEQ ID NO: 33 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A19. SEQ ID NO: 34 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A06. SEQ ID NO: 35 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A15. SEQ ID NO: 36 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A29. SEQ ID NO: 37 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B15. SEQ ID NO: 38 shows the amino acid sequence of a meningococcal serogroup A strain expressing factor H binding protein (fHBP) B16. SEQ ID NO: 39 shows the amino acid sequence of a meningococcal serogroup C strain expressing fHBP A10. SEQ ID NO: 39 also shows the amino acid sequence of a meningococcal serogroup W strain expressing fHBP A10. SEQ ID NO: 40 shows the amino acid sequence of a meningococcal serogroup W strain expressing fHBP A19. SEQ ID NO: 41 shows the amino acid sequence of a meningococcal serotype Y strain expressing fHBP B47. SEQ ID NO: 42 shows the amino acid sequence of a meningococcal serotype X strain expressing fHBP B49. SEQ ID NO: 43 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B44. SEQ ID NO: 44 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B09. SEQ ID NO: 45 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B09. SEQ ID NO: 46 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A05. SEQ ID NO: 47 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B01. SEQ ID NO: 48 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B01, including the N-terminal Cys at amino acid position 1. SEQ ID NO: 49 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B15, including the N-terminal Cys at amino acid position 1. SEQ ID NO: 50 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B16, including the N-terminal Cys at amino acid position 1. SEQ ID NO: 51 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B22. SEQ ID NO: 52 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A22. SEQ ID NO: 53 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A12. SEQ ID NO: 54 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A22. SEQ ID NO: 55 shows the amino acid sequence of N. meningitidis serotype B, 2086 variant A62, including the N-terminal Cys at amino acid position 1. SEQ ID NO: 56 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A62. SEQ ID NO: 57 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A29, including the N-terminal Cys at amino acid position 1. SEQ ID NO: 58 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B22. SEQ ID NO: 59 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A05. SEQ ID NO: 60 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A05. SEQ ID NO: 61 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B24. SEQ ID NO: 62 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B24. SEQ ID NO: 63 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A02. SEQ ID NO: 64 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A28. SEQ ID NO: 65 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A42. SEQ ID NO: 66 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A63. SEQ ID NO: 67 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant A76. SEQ ID NO: 68 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B05. SEQ ID NO: 69 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B07. SEQ ID NO: 70 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B08. SEQ ID NO: 71 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B13. SEQ ID NO: 72 shows the amino acid sequence of Neisseria meningitidis, serotype B, 2086 variant B52. SEQ ID NO: 73 shows the amino acid sequence of Neisseria meningitidis, serogroup B, 2086 mutant B107. SEQ ID NO: 74 shows the amino acid sequence of Neisseria meningitidis, serogroup B, 2086 mutant A56. DETAILED DESCRIPTION OF THE INVENTION

[0040] The inventors have found that compositions comprising at least one factor H binding protein (fHBP) and meningococcal serotypes A, C, W, and Y capsular glycoconjugates are stable and immunogenic. The inventors have further found that the compositions induce an immune response in humans against at least two, three, four, or five of meningococcal serotypes A, C, W, Y, and B. Surprisingly, the immune response elicited is higher than the immune response elicited by licensed vaccines against meningococcus.

[0041] In one aspect, a composition comprises at least one polypeptide derived from Neisseria meningitidis factor H binding protein and at least one Neisseria meningitidis capsular saccharide conjugated to a carrier protein. A protein derived from factor H binding protein (fHBP).

[0042] In one embodiment, the composition comprises any fHBP, such as, for example, any one of the following polypeptides: B24, B16, B44, A22, B03, B09, A12, A19, A05, A07, A06, A15, A29, B01, A62, B15, and any combination thereof. Preferably, the composition comprises a combination of A05 and B01 polypeptides. In another preferred embodiment, the composition comprises a B24 and A05 polypeptide combination. In another embodiment, the composition comprises an A05, A12, B09, and B44 polypeptide combination. In one embodiment, the composition comprises a lipidated fHBP. In one embodiment, the composition does not comprise a non-lipidated fHBP.

[0043] In another embodiment, the composition comprises a non-lipidated fHBP, such as any one of the non-lipidated fHBPs described in International Publication No. WO2012 / 032489, U.S. Patent Publication No. 20120093852, International Publication No. WO2013 / 132452, and U.S. Patent Publication No. 20160030543, each of which is incorporated by reference in its entirety. In one embodiment, the composition comprises at least one non-lipidated fHBP and at least one lipidated fHBP.

[0044] In some embodiments, the composition comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 , SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, and SEQ ID NO:62. Contains polypeptides with 9% identity.

[0045] In a preferred embodiment, the composition comprises: (a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2; (c) a meningococcal serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (d) a meningococcal serogroup A capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. (e) a meningococcal serogroup W capsular saccharide that is conjugated directly to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker; and (f) a meningococcal serogroup Y capsular saccharide that is conjugated directly to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker. The first polypeptide, MnB rLP2086 subfamily A (A05) protein In one embodiment, the composition comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. The polypeptide is a modified factor H binding protein (fHBP) from meningococcal strain M98250771. A description of fHBP is disclosed in WO2012032489 and U.S. Patent Publication No. 2012 / 0093852, each of which is incorporated by reference in its entirety. The polypeptide is N-terminally lipidated with three primary fatty acids, C16:0, C16:1, and C18:1, covalently attached at three positions of the polypeptide. The first polypeptide comprises a total of 258 amino acids.

[0046] A representative primary structure of the MnB rLP2086 A05 protein is presented in Figure 4 of U.S. Patent No. 10,183,070. The primary structure of the protein is illustrated in Figure 4 of U.S. Patent No. 10,183,070, using single-letter codes for all amino acids except the N-terminal cysteine ​​and glyceryl moieties (illustrated using the complete chemical formula). This structure includes the primary structure of the protein sequence, in which the N-terminal cysteine ​​residue is lipidated. The amino group of the N-terminal cysteine ​​residue at the protein N-terminus is attached to a fatty acid (R1) to form an amide bond, and the cysteinyl sulfhydryl group is attached to a glycerol moiety containing two ester-linked fatty acids (R2). The structure of R1 is predicted to be hexadecanoic acid (C16:0), while the structure of R2 varies depending on the MnB rLP2086 isoform.

[0047] The first polypeptide comprises two modifications introduced into the N-terminal region of the polypeptide compared to the corresponding wild-type sequence from meningococcal strain M98250771. A glycine at position 2 is added as a result of the introduction of a cloning site. The second modification comprises a deletion of four amino acids. Thus, in one embodiment, the first polypeptide comprises the CGSS sequence (SEQ ID NO: 3) at the N-terminus. See the first four amino acid residues of SEQ ID NO: 1.

[0048] The N-terminal differences between the first polypeptide sequence and the wild-type Neisseria sequence are shown below: Thus, in one embodiment, the first polypeptide comprises at least the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the first polypeptide comprises at least the first 4, more preferably at least the first 6, and most preferably at least the first 8 amino acid residues of SEQ ID NO: 1. Comparison of the recombinant and predicted N-terminal sequences of Neisseria subfamily A LP2086 polypeptides rLP2086 M98250771 CGSS----GGGGVAA D (SEQ ID NO: 4) Neisseria LP2086 M98250771 C-SSGS-GSGGGGVAAD (SEQ ID NO: 5) >A05 (SEQ ID NO: 1)

[0049] [ka]

[0050] In one embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the first polypeptide has a total of 258 amino acids. In one embodiment, the first polypeptide does not comprise an amino acid sequence having less than 100% sequence identity to SEQ ID NO: 1. In another embodiment, the first polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the first polypeptide comprises the amino acid sequence KDN. See, e.g., amino acid residues 73-75 of SEQ ID NO: 1. In another embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3 at the N-terminus of the polypeptide. In another embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 at the N-terminus of the polypeptide.

[0051] In a preferred embodiment, the first polypeptide is readily expressed in a recombinant host cell using standard techniques known in the art. In another preferred embodiment, the first polypeptide comprises a bactericidal epitope on the N and / or C domain of SEQ ID NO: 1. In one embodiment, the first polypeptide comprises at least the first 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 1 Preferably, the first polypeptide comprises at least the first two, more preferably at least the first four, and most preferably at least the first eight amino acid residues of SEQ ID NO:1.

[0052] In another aspect, the first polypeptide comprises at least the last 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues.

[0053] In one embodiment, the composition comprises about 30 μg / ml of a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the composition comprises about 60 μg of a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the composition comprises In another embodiment, the composition comprises about 60 μg of a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, and the composition preferably has a total volume of 0.5 ml. In another embodiment, the composition comprises about 120 μg / ml of a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. The second polypeptide, MnB rLP2086 subfamily B (B01) protein In one embodiment, the composition comprises a second polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The polypeptide is factor H binding protein (fHBP) from meningococcal strain CDC1573. A description of fHBP is disclosed in WO2012032489 and U.S. Patent Publication No. 2012 / 0093852, each of which is incorporated by reference in its entirety. The polypeptide is N-terminally lipidated with three primary fatty acids, C16:0, C16:1, and C18:1, covalently attached at three positions of the polypeptide. The second polypeptide comprises a total of 261 amino acids.

[0054] A representative primary structure of the MnB rLP2086 B01 protein is presented in Figure 5 of U.S. Patent No. 10,183,070. The primary structure of the protein is illustrated in Figure 5 of U.S. Patent No. 10,183,070, using single-letter codes for all amino acids except the N-terminal cysteine ​​and glyceryl moieties (illustrated using the complete chemical formula). This structure includes the primary structure of the protein sequence, in which the N-terminal cysteine ​​residue is lipidated. The amino group of the N-terminal cysteine ​​residue at the protein N-terminus is attached to a fatty acid (R1) to form an amide bond, and the cysteinyl sulfhydryl group is attached to a glycerol moiety containing two ester-linked fatty acids (R2). The structure of R1 is predicted to be hexadecanoic acid (C16:0), while the structure of R2 varies depending on the rLP2086 isoform.

[0055] The second polypeptide contains one modification introduced within the N-terminal region of the rLP2086 subfamily B protein compared to the corresponding wild-type sequence from meningococcal strain CDC-1573: a glycine at position 2 is the result of introducing a cloning site.

[0056] The N-terminal differences from the original Neisserial sequence are shown below. Comparison of the predicted N-terminal sequences of recombinant and Neisserial subfamily B LP2086 proteins rLP2086 CDC-1573 CGSSGGGGSGGGGVTAD (SEQ ID NO: 24) Neisseria LP2086 CDC-1573 C-SSGGGGSGGGGVTAD (SEQ ID NO: 25) In one embodiment, the second polypeptide comprises a CGSS sequence (SEQ ID NO:3) at its N-terminus. See the first four amino acid residues of SEQ ID NO:2. >B01 (SEQ ID NO: 2)

[0057] [ka]

[0058] In one embodiment, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the second polypeptide has a total of 261 amino acids. In one embodiment, the second polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the second polypeptide does not include a polypeptide having less than 100% sequence identity with SEQ ID NO: 2. Preferred In embodiments, the first polypeptide and the second polypeptide comprise a CGSS (SEQ ID NO: 3) sequence at the N-terminus of each polypeptide.

[0059] In a preferred embodiment, the second polypeptide is readily expressed in a recombinant host cell using standard techniques known in the art. In another preferred embodiment, the second polypeptide comprises a bactericidal epitope on the N and / or C domain of SEQ ID NO: 2. In one embodiment, the second polypeptide comprises at least the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues of SEQ ID NO:2. Preferably, the second polypeptide comprises at least the first two, more preferably at least the first four, and most preferably at least the first eight amino acid residues of SEQ ID NO:2.

[0060] In another embodiment, the second polypeptide comprises at least the last 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 of the amino acid sequence set forth in SEQ ID NO:2. , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues.

[0061] In one embodiment, the composition comprises about 30 μg / ml of a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. In a preferred embodiment, the composition comprises about 60 μg of a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. In a preferred embodiment, the composition comprises about 60 μg of a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, and the composition preferably has a total volume of 0.5 ml. In another embodiment, the composition comprises 120 μg / ml of a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. Meningococcal serogroups A, C, W, and Y (MenACWY) capsular saccharides Throughout this specification, the term "saccharide" may refer to polysaccharides or oligosaccharides, or may include both. Polysaccharides are isolated from bacteria, or isolated from bacteria and sized to some extent by known methods, and optionally by microfluidization. Polysaccharides can be sized to reduce viscosity in polysaccharide samples and / or to improve filterability of the conjugate product. Oligosaccharides have a low number of repeating units (typically 5-30 repeating units) and are typically hydrolyzed polysaccharides.

[0062] Each meningococcal capsular saccharide may be conjugated to a carrier protein independently selected from the group consisting of TT, DT, CRM197, fragment C of TT, and protein D. One or more meningococcal capsular saccharides may be conjugated to a different carrier protein from the others, although in one aspect they are all conjugated to the same carrier protein. For example they may be conjugated to the same carrier protein selected from the group consisting of TT, DT, CRM197, fragment C of TT, and protein D. In this context, CRM197 and DT may be considered to be the same carrier protein as they differ by only one amino acid. In a preferred aspect, all meningococcal capsular saccharides present are conjugated to a carrier protein independently selected from the group consisting of TT, DT, CRM197, fragment C of TT, and protein D. All meningococcal capsular saccharides are conjugated to TT.

[0063] Where the protein carrier is the same for two or more sugars in the composition, the sugars may be conjugated to the same molecule of protein carrier (with two or more different sugars conjugated to the carrier molecule) (see, e.g., WO 04 / 083251). For example, a single carrier protein may be conjugated to MenA and MenC; MenA and MenW; MenA and MenY; MenC and MenW; MenC and MenY; MenW and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MenY; MenC, MenW and MenY; MenA, MenC, MenW and MenY. Alternatively, the sugars may each be separately conjugated to a different molecule of protein carrier (with only one type of sugar conjugated to each molecule of protein carrier).

[0064] In one embodiment, at least two different glycoconjugates are separately conjugated to the same type of carrier protein, where one or more sugars are conjugated to the carrier protein via a first type of chemical group on the protein carrier and one or more sugars are conjugated to the carrier protein via a second (different) type of chemical group on the protein carrier.

[0065] In one embodiment, the two conjugates comprise the same sugar linked to the same carrier but by different conjugation chemistries, hi an alternative embodiment, the two different sugars are conjugated to different groups on the protein carrier.

[0066] "Separately conjugated to the same type of carrier protein" means that the saccharides are individually conjugated to the same carrier (e.g., MenA is conjugated to tetanus toxoid via an amine group on tetanus toxoid, and MenC is conjugated to tetanus toxoid via a carboxylic acid group on a different molecule of tetanus toxoid). The capsular saccharides may be conjugated to the same carrier protein independently selected from the group consisting of TT, DT, CRM197, fragment C of TT, and protein D. A more complete list of protein carriers that may be used in the conjugates of the disclosure is provided below. In this context, CRM197 and DT may be considered to be the same carrier protein as they differ by only one amino acid. In one aspect, all capsular saccharides present are conjugated to TT.

[0067] The saccharide comprises any one of a meningococcal serogroup A capsular saccharide (MenA), a meningococcal serogroup C capsular saccharide (MenC), a meningococcal serogroup Y capsular saccharide (MenY), and a meningococcal serogroup W capsular saccharide (MenW), or any combination thereof.

[0068] The first and second chemical groups present on the protein carrier are different from each other, and ideally, are natural chemical groups that can be easily used for conjugation purposes.They can be independently selected from the group consisting of carboxyl group, amino group, sulfhydryl group, hydroxyl group, imidazolyl group, guanidyl group, and indolyl group.In one embodiment, the first chemical group is carboxyl and the second is amino, or vice versa.These groups are described in detail below.

[0069] In a specific aspect, the immunogenic composition comprises at least two different meningococcal capsular saccharides, where one or more are selected from a first group consisting of MenA and MenC, conjugated to a carrier protein via a first type of chemical group (e.g. carboxyl) on the protein carrier, and one or more different saccharides are conjugated to a carrier protein via a second type of chemical group (e.g. carboxyl) on the protein carrier. The antibody is selected from a second group consisting of MenC, MenY, and MenW, conjugated to a carrier protein via a chemical group (e.g., amino) of the same type.

[0070] In a further aspect, the immunogenic compositions of the disclosure comprise MenA conjugated via a first type of chemical group (e.g., carboxyl) and MenC conjugated via a second type of chemical group (e.g., amino).

[0071] In another embodiment, the immunogenic composition comprises MenC conjugated via a first type of chemical group (e.g., carboxyl) and MenY conjugated via a second type of chemical group (e.g., amino).

[0072] In another embodiment, the immunogenic composition comprises MenA conjugated via a first type of chemical group (e.g., carboxyl) and MenC, MenY, and MenW conjugated via a second type of chemical group (e.g., amino).

[0073] In another embodiment, the immunogenic composition comprises MenA and MenC conjugated via a first type of chemical group (e.g., carboxyl) and MenY and MenW conjugated via a second type of chemical group (e.g., amino).

[0074] The saccharide of the present disclosure included in the pharmaceutical (immunogenic) composition of the present disclosure may be tetanus toxoid (TT), tetanus toxoid fragment C, a non-toxic mutant of tetanus toxin (note that for the purposes of this disclosure, all such mutants of TT are considered to be the same type of carrier protein), diphtheria toxoid (DT), CRM197, other non-toxic mutants of diphtheria toxin [CRM176, CRM197, CRM228, CRM45 (Uchida et al., J. Biol. Chem., Vol. 218; pp. 3838-3844, 1973); CRM9, CRM45, CRM102, CRM103, and CRM107, and Nicholls and Youle, Genetically Engineered Toxins, edited by Frankel, Maecel Dekker Inc., 1992; deletions or mutations of Glu-148 to Asp, Gln, or Ser and / or Ala158 to Gly, and other mutations disclosed in US4709017 or US4950740; mutations of at least one or more residues Lys516, Lys526, Phe530, and / or Lys534, and other mutations disclosed in US5917017 or US6455673; or fragments disclosed in US5843711, etc. (note that for the purposes of this disclosure, all such variants of DT are considered to be the same type of carrier protein), pneumococcal pneumolysin (Kuo et al., (1995) Infect. Immun, vol. 63; pp. 2706-13), OMPC (meningococcal outer membrane protein - usually extracted from meningococcal serogroup B - EP0372501), synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO93 / 17712, WO94 / 03208), pertussis proteins (WO98 / 58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO91 / 01146), N19 proteins (Baraldoi et al., (2004) Infect Immun, 72; 4884-4887), artificial proteins containing multiple human CD4+ T cell epitopes from antigens derived from various pathogens, such as the pneumococcal surface protein PspA (WO 02 / 091998) (Falugi et al., (2001) Eur J Immunol, 31; 3816-3824), iron uptake protein (WO 01 / 72337), C. difficile toxin A or B (WO 00 / 61761), or protein D (EP 594610 and WO 00 / 56360).

[0075] In one aspect, an immunogenic composition of the disclosure uses (independently) the same type of carrier protein for at least two, three, four, or each saccharide contained therein. In one aspect the immunogenic composition of the disclosure comprises a meningococcal saccharide conjugated to a carrier protein selected from the group consisting of TT, DT, CRM197, fragment C of TT, and protein D.

[0076] Immunogenic compositions of the disclosure optionally comprise at least one meningococcal saccharide (e.g. MenA; MenC; MenW; MenY; MenA and MenC; MenA and MenW; MenA and MenY; MenC and MenW; MenC and MenY; MenW and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MenY; MenC, MenW and MenY; or MenA, MenC, MenW and MenY) conjugate, in a ratio of Men saccharide to carrier protein of 1:5 to 5:1, 1:2 to 5:1, 1:0.5 to 1:2.5, or 1:1.25 to 1:2.5 (w / w). In one preferred embodiment, the composition comprises MenA, MenC, MenW, and MenY, each conjugated to tetanus toxoid at ratios (toxoid to polysaccharide) of about 3, about 3, about 1.5, and about 1.3, respectively.

[0077] The sugar to carrier protein ratio (w / w) in the conjugate can be determined using sterile conjugates: the amount of protein is determined using the Lowry assay (e.g., Lowry et al., (1951) J. Biol. Chem., 193:265-275 or Peterson et al., Analytical Biochemistry, 100:201-220 (1979)), and the amount of sugar is determined using ICP-OES (inductively coupled plasma-optical emission spectroscopy) for MenA, DMAP assay for MenC, and resorcinol assay for MenW and MenY (Monsigny et al., (1988) Anal. Biochem., 175:525-530).

[0078] In one aspect, the immunogenic composition of the disclosure comprises a meningococcal saccharide conjugate, wherein the meningococcal saccharide is conjugated to a carrier protein via a linker, for example a bifunctional linker. The linker is optionally heterobifunctional or homobifunctional, for example having a reactive amino group and a reactive carboxylic acid group, two reactive amino groups, or two reactive carboxylic acid groups. The linker has, for example, 4 to 20, 4 to 12, or 5 to 10 carbon atoms. A possible linker is ADH. Other linkers include β-propionamide (WO 00 / 10599), nitrophenyl-ethylamine (Gever et al. (1979) Med. Microbiol. Immunol., 165:171-288), haloalkyl halides (US 4,057,685), glycosidic bonds (US 4,673,574, US 4,808,700), hexanediamine, and 6-aminocaproic acid (US 4,459,286).

[0079] The sugar conjugates present in the immunogenic compositions of the present disclosure may be prepared by any known coupling technique. Conjugation methods may rely on activating sugars to form cyanate esters using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). The activated sugars may then be coupled to amino groups on carrier proteins, either directly or via a spacer (linker) group. For example, the spacer may be cystamine or cysteamine, resulting in a thiolated polysaccharide that can be coupled to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using GMBS) or a holoacetylated carrier protein (e.g., using iodoacetimide or N-succinimidyl bromoacetate). Optionally, the cyanate ester (optionally generated by CDAP chemistry) may be coupled with hexanediamine or ADH to form the acylate. The amino-derivatized sugars are conjugated to carrier proteins via carboxyl groups on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry. Such conjugates are described in Uniformed Services University PCT published applications WO 93 / 15760, as well as WO 95 / 08348 and WO 96 / 29094.

[0080] Other suitable techniques use carbiinides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in WO 98 / 42721. Conjugation can involve a carbonyl linker, which can be formed by reaction of the free hydroxyl group of the sugar with CDI (Bethell et al., J. Biol. Chem., 1979, vol. 254; pp. 2572-2574; Hearn et al., J. Chromatogr., 1981, vol. 218; pp. 509-18), followed by reaction with the protein to form a carbamate bond. This can involve reduction of the anomeric terminus to a primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and optional protection / deprotection of the primary hydroxyl group, followed by coupling of the CDI carbamate intermediate to an amino group on the protein.

[0081] Conjugates can also be prepared by direct reductive amination methods as described in US 4,365,170 (Jennings) and US 4,673,574 (Anderson). Other methods are described in EP-0-161-188, EP-208,375, and EP-0-477,508.

[0082] A further method involves coupling of adipic acid hydrazide (ADH)-derivatized cyanogen bromide (or CDAP)-activated sugars to protein carriers by carbodiimide condensation (Chu C. et al., Infect. Immunity, 1983, pp. 245-256), e.g., using EDAC.

[0083] In one aspect, a hydroxyl group on the saccharide (optionally an activated hydroxyl group, for example a hydroxyl group activated by a cyanate ester) is linked, either directly or indirectly (via a linker), to an amino or carboxyl group on the protein. If a linker is present, the hydroxyl group on the saccharide is optionally linked to an amino group on the linker, for example by using CDAP conjugation. A further amino group in the linker, for example ADH), may be conjugated to a carboxylic acid group on the protein, for example by using carbodiimide chemistry, for example by using EDAC. In one aspect, the meningococcal capsular saccharide (or saccharide in general) is first conjugated to the linker before the linker is conjugated to the carrier protein. Alternatively, the linker may be conjugated to the carrier before being conjugated to the saccharide.

[0084] Generally, the following types of chemical groups on protein carriers can be used for coupling / conjugation: A) Carboxyl (e.g., via aspartic acid or glutamic acid). In one embodiment, this group is linked to an amino group on the sugar directly or to an amino group on the linker using carbodiimide chemistry, e.g., EDAC. B) Amino groups (e.g., via lysine). In one embodiment, this group is linked directly to a carboxyl group on the sugar or to a carboxyl group on the linker using carbodiimide chemistry, e.g., EDAC. In another embodiment, this group is linked directly to a CDAP- or CNBr-activated hydroxyl group on the sugar or to such a group on the linker, a sugar or linker bearing an aldehyde group, or a sugar or linker bearing a succinimide ester group. C) Sulfhydryl (e.g., via cysteine). In one embodiment, this group is linked to a bromo- or chloroacetylated sugar or linker with maleimide chemistry. In one embodiment, this group is activated / modified with bisdiazobenzidine. D) A hydroxyl group (e.g., via tyrosine). In one embodiment, this group is activated / modified with bisdiazobenzidine. E) Imidazolyl group (e.g., via histidine). In one embodiment, this group is activated / modified with bisdiazobenzidine. F) A guanidyl group (e.g., via arginine). G) Indolyl groups (e.g., via tryptophan).

[0085] On the sugar, the following groups are generally available for coupling: OH, COOH, or NH2. Aldehyde groups can be generated after various treatments known in the art, such as periodate, acid hydrolysis, and hydrogen peroxide. Direct Coupling Method: Sugar-OH + CNBr or CDAP → Cyanate ester + NH2-Prot → Conjugate Sugar-aldehyde + NH2-Prot → Schiff base + NaCNBH3 → conjugate Sugar-COOH+NH2-Prot+EDAC → conjugate Sugar-NH2+COOH-Prot+EDAC → conjugate Indirect coupling via a spacer (linker): Sugar-OH + CNBr or CDAP → Cyanate ester + NH2-NH2 → Sugar-NH2 + COOH-Prot + EDAC → Conjugate Sugar-OH + CNBr or CDAP → cyanate ester + NH2-SH → Sugar-SH + SH-Prot (obtained from a native protein with exposed cysteines or after modification of the amino groups of a protein, for example, by SPDP) → Sugar-SS-Prot Sugar-OH + CNBr or CDAP → Cyanate ester + NH2-SH → Sugar-SH + Maleimide-Prot (amino group modification) → Conjugate Sugar-COOH+EDAC+NH2-NH2 → Sugar-NH2+EDAC+COOH-Prot → Conjugate Sugar-COOH + EDAC + NH2-SH → Sugar-SH + SH-Prot (native protein with exposed cysteines or obtained after modification of the amino groups of the protein, for example, by SPDP) → Sugar-SS-Prot Sugar-COOH + EDAC + NH2-SH → Sugar-SH + Maleimide-Prot (amino group modification) → Conjugate Sugar-aldehyde + NH2-NH2 → Sugar-NH2 + EDAC + COOH-Prot → Conjugate Note: Any suitable carbodiimide may be substituted for EDAC above.

[0086] In summary, the types of protein carrier chemical groups that can generally be used for coupling to sugars are amino groups (e.g., on lysine residues), COOH groups (e.g., on aspartic acid and glutamic acid residues), and SH groups (where accessible) (e.g., on cysteine ​​residues).

[0087] In one aspect at least one of the meningococcal capsular saccharides (or saccharides in general) is directly conjugated to a carrier protein, optionally MenW and / or MenY and / or MenC saccharides are directly conjugated to a carrier protein, for example MenW; MenY; MenC; MenW and MenY; MenW and MenC; MenY and MenC; or MenW, MenY and MenC are directly linked to a carrier protein. Optionally at least one of the meningococcal capsular saccharides is directly conjugated by CDAP, for example MenW, MenY, MenC, MenW and Men Y, MenW and MenC, MenY and MenC, or MenW, MenY and MenC are directly linked to a carrier protein by CDAP (see WO95 / 08348 and WO96 / 29094). In one aspect, all meningococcal capsular saccharides are conjugated to tetanus toxoid.

[0088] In one aspect, the ratio of MenW and / or Y sugars to carrier protein is 1:0.5 to 1:2 (w / w), and / or the ratio of MenC sugars to carrier protein is 1:0.5 to 1:4 or 1:0.5 to 1:1.5 (w / w), particularly when these sugars are optionally directly linked to the protein using CDAP.

[0089] In one aspect, at least one of the meningococcal capsular saccharides (or saccharides in general) is conjugated to the carrier protein via a linker, for example a bifunctional linker. The linker is optionally heterobifunctional or homobifunctional, for example having a reactive amine group and a reactive carboxylic acid group, two reactive amine groups, or two reactive carboxylic acid groups. The linker has, for example, 4 to 20, 4 to 12, 5 to 10 carbon atoms. A possible linker is ADH.

[0090] In one embodiment, MenA; MenC; or MenA and MenC are conjugated to a carrier protein (eg, tetanus toxoid) via a linker. In one aspect, at least one meningococcal saccharide is conjugated to a carrier protein via a linker using CDAP and EDAC. For example, MenA, MenC, or MenA and MenC are conjugated to the protein via a linker (e.g. one having two hydrazino groups at its termini, such as ADH) using CDAP and EDAC as described above. For example, CDAP is used to conjugate the saccharide to the linker and EDAC is used to conjugate the linker to the protein. Optionally, conjugation via the linker results in a saccharide to carrier protein ratio of 1:0.5 to 1:6, 1:1 to 1:5, or 1:2 to 1:4 for MenA; MenC; or MenA and MenC.

[0091] In one aspect, the MenA capsular saccharide, if present, is at least partially O-acetylated such that at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeating units are O-acetylated at at least one position, for example O-acetylation is present at at least the O-3 position of at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeating units.

[0092] In one aspect, the MenC capsular saccharide, if present, is at least partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the (α2→9)-linked NeuNAc repeat units are O-acetylated at at least one or two positions, e.g., O-acetylation is present at the O-7 and / or O-8 positions of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.

[0093] In one aspect, the MenW capsular saccharide, if present, is at least partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeating units are O-acetylated at at least one or two positions, for example O-acetylation is present at the O-7 and / or O-9 positions of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeating units.

[0094] In one aspect, the MenY capsular saccharide, if present, comprises at least 20%, 30% of the repeating units. At least partially O-acetylated such that at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the repeat units are O-acetylated at at least one or two positions. O-acetylation is present at the 7 and / or 9 positions of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the repeat units.

[0095] Percent O-acetylation refers to the percentage of repeat units that contain O-acetylation, which can be measured on the saccharide before and / or after conjugation.

[0096] In one aspect of the disclosed immunogenic composition the, or each meningococcal capsular saccharide present is conjugated to TT. In a further aspect each meningococcal capsular saccharide is separately conjugated to a separate carrier protein. In a further aspect each meningococcal capsular saccharide conjugate has a saccharide:carrier ratio of 1:5 to 5:1 or 1:1 to 1:4 (w / w). In a further aspect at least one, two or three meningococcal capsular saccharide conjugates are directly conjugated to a carrier protein. In a further aspect MenW and / or MenY, MenW and / or MenC, MenY and / or MenC, or MenW, MenC and MenY are directly conjugated to a carrier protein. In a further aspect at least one, two or three meningococcal saccharide conjugates are directly conjugated by CDAP chemistry. In a further aspect, the ratio of MenW and / or Y saccharide to carrier protein is 1:0.5 to 1:2 (w / w). In a further aspect, the ratio of MenC saccharide to carrier protein is 1:0.5 to 1:2 (w / w). In a further aspect, at least one, two or three meningococcal capsular saccharides are conjugated to the carrier protein via a linker (which may have two reactive amino groups (such as ADH) or two reactive carboxyl groups, or may be bifunctional, such as having a reactive amino group at one terminus and a reactive carboxyl group at the other terminus). The linker may have 4 to 12 carbon atoms. In a further aspect, the or each meningococcal capsular saccharide conjugated via a linker is conjugated to the linker using CDAP chemistry. In a further aspect, the carrier protein is conjugated to the linker using carbodiimide chemistry, for example using EDAC. In a further aspect, the or each meningococcal capsular saccharide is conjugated to a linker before the carrier protein is conjugated to the linker, hi a further aspect, MenA is conjugated to the carrier protein via a linker (the ratio of MenA saccharide to carrier protein may be 1:2 to 1:5 (w / w)).In a further embodiment, the MenC is conjugated to a carrier protein via a linker (the ratio of MenC saccharide to carrier protein may be 1:2 to 1:5 (w / w)).

[0097] The use of native or slightly sized polysaccharide conjugates can achieve one or more of the following advantages: 1) highly immunogenic conjugates that can be filtered through a 0.2 micron filter; 2) enhanced immunological memory (as in Example 3); 3) altering the polysaccharide-to-protein ratio in the conjugate, such that the polysaccharide-to-protein ratio (w / w) in the conjugate can be increased (which may result in a reduced carrier suppression effect); and 4) using larger polysaccharides in the conjugation can stabilize immunogenic conjugates prone to hydrolysis (such as MenA conjugates). The use of larger polysaccharides may result in more cross-linking with the conjugate carrier, reducing the release of free sugar from the conjugate. Conjugate vaccines described in the prior art tend to depolymerize the polysaccharide prior to conjugation to improve conjugation. Meningococcal (or saccharide) conjugate vaccines that retain larger sized saccharides may provide a better immune response against meningococcal disease.

[0098] Thus, the immunogenic compositions of the present disclosure may comprise one or more saccharide conjugates, wherein the average size of each saccharide prior to conjugation is greater than 50 kDa, 75 kDa, 100 kDa, 110 kDa, 120 kDa, or 130 kDa. In one aspect, the conjugate following conjugation should be readily filterable through a 0.2 micron filter, such that a yield of greater than 50, 60, 70, 80, 90, or 95% is obtained after filtration compared to the pre-filtration sample.

[0099] In particular, the immunogenic compositions of the disclosure comprise meningococcal capsular saccharides from at least one, two, three or four of serogroups A, C, W and Y conjugated to a carrier protein, wherein at least one, two, three or four, or each, of the meningococcal saccharides has an average size (weight average molecular weight, Mw) of greater than 50kDa, 60kDa, 75kDa, 100kDa, 110kDa, 120kDa or 130kDa.

[0100] In a preferred embodiment, MenA AH The average Mw of the MenC-TT conjugate is at least 250 kDa, 260 kDa, 270 kDa, 280 kDa, or 290 kDa, and most preferably about 300 kDa, and up to 350 kDa or 330 kDa. AH The average Mw of the MenW-TT conjugate is at least 150 kDa, 160 kDa, 170 kDa, 180 kDa, or 190 kDa, most preferably about 200 kDa, and up to 250 kDa or 230 kDa. In preferred embodiments, the average Mw of the MenW-TT conjugate is at least 240, 250 kDa, 260 kDa, or 270 kDa, most preferably about 280 kDa, and up to 330 kDa or 310 kDa. In preferred embodiments, the average Mw of the MenY-TT conjugate is at least 220 kDa, 230 kDa, 240 kDa, or 250 kDa, most preferably about 270 kDa, and up to 320 kDa or 300 kDa.

[0101] The immunogenic composition may comprise meningococcal capsular saccharides from at least one, two, three or four of serotypes A, C, W and Y conjugated to a carrier protein, wherein at least one, two, three or four, or each, meningococcal saccharide is either a native saccharide or is sized by a factor of x2, x3, x4, x5, x6, x7, x8, x9 or up to x10 compared to the weight average molecular weight of the native polysaccharide.

[0102] For purposes of this disclosure, "native polysaccharide" refers to a saccharide that has not been subjected to a process whose purpose is to reduce the size of the saccharide. A polysaccharide may be slightly reduced in size during normal purification procedures. Such a saccharide is still native. Only if the polysaccharide has been subjected to sizing techniques is the polysaccharide considered non-native.

[0103] For purposes of this disclosure, "sizing by a factor of up to x2" means subjecting the sugar to a process intended to reduce the size of the sugar but retain a size greater than half the size of the native polysaccharide. x3, x4, etc. are to be interpreted similarly, i.e., subjecting the sugar to a process intended to reduce the size of the polysaccharide but retain a size greater than one-third, one-quarter, etc. the size of the native polysaccharide.

[0104] In one aspect of the disclosure, an immunogenic composition comprises meningococcal capsular saccharides from at least one, two, three or four of serogroups A, C, W and Y conjugated to a carrier protein, wherein at least one, two, three or four, or each, of the meningococcal saccharides is a native polysaccharide.

[0105] In one aspect of the disclosure an immunogenic composition comprises meningococcal capsular saccharides from at least one, two, three or four of serotypes A, C, W and Y conjugated to a carrier protein, wherein at least one, two, three or four, or each, of the meningococcal saccharides is sized by a factor of x 1.5, x 2, x 3, x 4, x 5, x 6, x 7, x 8, x 9 or up to x 10.

[0106] The immunogenic compositions of the disclosure optionally comprise the following conjugates: meningococcal serogroup C capsular saccharide (MenC), serogroup A capsular saccharide (MenA), serogroup W135 capsular saccharide (MenW), serogroup Y capsular saccharide (MenY), serogroups C and Y capsular saccharide (MenCY), serogroups C and A capsular saccharide (MenAC), serogroups C and W capsular saccharide (MenCW), serogroups A and Y capsular saccharide ( and Y capsular saccharide (MenA, W, Y), serotypes A, C, and W capsular saccharide (MenA, W, Y), serotypes A, C, and Y capsular saccharide (MenA, W, Y), serotypes C, W, Y, and Y capsular saccharide (MenC, W, Y), or serotypes A, C, W, Y, and Y capsular saccharide (MenA, W, Y). This is the definition of "one, two, three, or four" or "at least one" of serotypes A, C, W, and Y, or each meningococcal saccharide when referred to herein.

[0107] In one aspect the average size of at least one, two, three, four or each meningococcal saccharide is between 50KDa and 1500kDa, 50kDa and 500kDa, 50kDa and 300KDa, 101kDa and 1500kDa, 101kDa and 500kDa, 101kDa and 300kDa, as determined by MALLS.

[0108] In one aspect the MenA saccharide, if present, has a molecular weight of 50-500kDa, 50-100kDa, 100-500kDa, 55-90KDa, 60-70kDa, or 70-80kDa, or 60-80kDa.

[0109] In one aspect the MenC saccharide, if present, has a molecular weight of 100-200 kDa, 50-100 kDa, 100-150 kDa, 101-130 kDa, 150-210 kDa, or 180-210 kDa.

[0110] In one aspect, the MenY saccharide, if present, has a molecular weight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa, or 110-140 kDa, 50-100 kDa, 100-140 kDa, 140-170 kDa, or 150-160 kDa.

[0111] In one aspect, the MenW sugar, if present, has a molecular weight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa, 110-140 kDa, 50-100 kDa, or 120-140 kDa.

[0112] As used herein, the molecular weight or average molecular weight of a saccharide refers to the weight average molecular weight (Mw) of the saccharide measured prior to conjugation, as measured by MALLS. The MALLS technique is well known in the art and is typically performed as described in Example 2. MALLS analysis of meningococcal saccharides may use a combination of two columns (TSKG6000 and 5000PWx1) and saccharides are eluted in water. Sugars are detected using a light scattering detector (e.g., a Wyatt Dawn DSP equipped with a 10mW argon laser, 488nm) and an interference refractometer (e.g., a Wyatt Otilab DSP equipped with a P100 cell and a 498nm red filter).

[0113] In one aspect the meningococcal saccharide is a native polysaccharide or a native polysaccharide that has been reduced in size during conventional extraction processes. In one aspect the meningococcal saccharide is sized by mechanical disruption, for example by microfluidisation or sonication. Microfluidisation and sonication have the advantage of reducing the size of larger native polysaccharides sufficiently to provide a filterable conjugate (e.g. pass through a 0.2 micron filter). Sizing is by a factor of less than x20, x10, x8, x6, x5, x4, x3, x2 or x1.5.

[0114] In one aspect, the immunogenic composition comprises a meningococcal conjugate made from a mixture of native polysaccharides and saccharides sized by a factor of less than x20. For example, the saccharides from MenC and / or MenA are native. For example, the saccharides from MenY and / or MenW are sized by a factor of less than x20, x10, x8, x6, x5, x4, x3, or x2. For example, the immunogenic composition contains a conjugate made from MenY and / or MenW and / or MenC and / or MenA sized and / or microfluidized by a factor of less than x10. For example, the immunogenic composition contains a conjugate made from native MenA and / or MenC and / or MenW and / or MenY. For example, the immunogenic composition includes a conjugate made from native MenC. For example, the immunogenic composition includes a conjugate made from native MenC and MenA sized and / or microfluidized by a factor of less than x10. For example, the immunogenic composition includes a conjugate made from native MenC and MenY that has been sized and / or microfluidized by a factor of less than x10.

[0115] In one aspect, the polydispersity of the saccharide is 1-1.5, 1-1.3, 1-1.2, 1-1.1, or 1-1.05, and after conjugation to the carrier protein, the polydispersity of the conjugate is 1.0-2.5, 1.0-2.0, 1.0-1.5, 1.0-1.2, 1.5-2.5, 1.7-2.2, or 1.5-2.0. All polydispersity measurements are by MALLS.

[0116] The saccharide is optionally sized up to 1.5, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 times the size of the polysaccharide isolated from the bacteria. In one aspect each meningococcal saccharide is either a native polysaccharide or sized by a factor of less than x10. In a further aspect each meningococcal capsular saccharide is a native polysaccharide. In a further aspect at least one, two, three or four meningococcal capsular saccharides are sized by microfluidisation. In a further aspect each meningococcal capsular saccharide is sized by a factor of less than x10. In a further aspect the meningococcal conjugate is made from a mixture of native polysaccharide and saccharides sized by a factor of less than x10. In a further aspect the capsular saccharide from serotype Y is sized by a factor of less than x10. In a further aspect the capsular saccharides from serotypes A and C are native polysaccharides and saccharides from serotypes W135 and Y are sized by a factor of less than x10. In a further aspect the average size of each meningococcal capsular saccharide is between 50kDa and 300kDa or between 50kDa and 200kDa, In a further aspect the immunogenic composition comprises MenA capsular saccharides having an average size of more than 50kDa, 75kDa, 100kDa or an average size of 50-100kDa or 55-90kDa or 60-80kDa. In a further aspect, the immunogenic composition comprises MenC capsular saccharides having an average size of more than 50kDa, 75kDa, 100kDa, or between 100 and 200kDa, 100 and 150kDa, 80 and 120kDa, 90 and 110kDa, 150 and 200kDa, 120 and 240kDa, 140 and 220kDa, 160 and 200kDa, or 190 and 200kDa. In a further aspect the immunogenic composition comprises MenY capsular saccharides having an average size of more than 50kDa, 75kDa, 100kDa, or 60-190kDa or 70-180kDa or 80-170kDa or 90-160kDa or 100-150kDa, 110-145kDa or 120-140kDa. The composition comprises MenW capsular saccharides having an average size of more than 50kDa, 75kDa, 100kDa, or 60-190kDa or 70-180kDa or 80-170kDa or 90-160kDa or 100-150kDa, 140-180kDa, 150-170kDa or 110-140kDa.

[0117] In one aspect of the disclosure, the saccharide dose of each of the at least two, three, four, or each of the meningococcal saccharide conjugates is optionally the same or approximately the same. In one aspect, the immunogenic compositions of the disclosure are adjusted or buffered to a pH of 7.0-8.0, a pH of 7.2-7.6, or at or about pH 7.4.

[0118] The immunogenic compositions or vaccines of the present disclosure are optionally lyophilized in the presence of a stabilizer, for example, a polyol such as sucrose or trehalose. In the meningococcal saccharide combinations mentioned above, it may be advantageous to use any aluminium salt adjuvant or no adjuvant at all.

[0119] The active agent can be present in various concentrations in the pharmaceutical composition or vaccine of the present disclosure. Typically, the minimum concentration of a substance is the amount necessary to achieve its intended use, while the maximum concentration is the maximum amount that can be kept in solution or homogeneously suspended in the initial mixture. For example, the minimum amount of a therapeutic agent is optionally that which provides a single therapeutically effective dosage. For a biologically active substance, the minimum concentration is the amount required for biological activity upon reconstitution, and the maximum concentration is the point at which a homogeneous suspension cannot be maintained.

[0120] In another embodiment, the composition comprises a conjugate of meningococcal serogroup X capsular polysaccharide and a carrier molecule. The structure of the type X capsular polysaccharide consists of N-acetylglucosamine-4-phosphate residues held together by al-4 phosphodiester bonds that do not contain O-acetyl groups. The carrier molecule can be diphtheria or tetanus toxoid, CRM197, or protein D. In a preferred embodiment, as illustrated in the Examples, the composition does not comprise a conjugate of meningococcal serogroup X capsular polysaccharide.

[0121] Further description of exemplary compositions is provided below. Compositions and vaccines In some embodiments, the composition comprises a lyophilized MenACWY-TT composition that is reconstituted with a liquid MnB divalent rLP2086 composition. The lyophilized MenACWY-TT composition and the liquid MnB divalent rLP2086 composition are preferably compatible and stable at room temperature for at least 24 hours after reconstitution.

[0122] In preferred embodiments, the compositions induce bactericidal antibodies against meningococcal serotype B and meningococcal serotypes other than B. For example, in some embodiments, MnB bivalent rLP2086 compositions induce bactericidal antibodies against at least meningococcal serotypes A, C, W, Y, and X.

[0123] Furthermore, the inventors found that the composition induced geometric mean titers against meningococcal serotypes A, C, W, and Y that were consistent with those observed with licensed vaccines against meningococcal serotypes A, C, W, and Y.

[0124] In some embodiments, the composition elicits geometric mean titers against meningococcal serotypes A, C, W, and Y that are higher than the geometric mean titers observed with licensed vaccines against meningococcal serotypes A, C, W, and Y.

[0125] Furthermore, the inventors have demonstrated that the composition exhibits the same protective effect as observed in licensed vaccines against meningococcal serogroup B. We found that the immunizations induced geometric mean titers against N. meningitidis serogroup B that were consistent with the geometric mean titers obtained from the immunizations.

[0126] In some embodiments, the composition elicits a geometric mean titer against meningococcal serogroup B that is higher than the geometric mean titer observed with a licensed vaccine against meningococcal serogroup B. In some embodiments, compositions including fHBP induce an effective immune response in humans at least 12 months of age. The compositions also induce an immune response against meningococcal serogroup X strains. In some embodiments, the compositions comprise at least one factor H binding polypeptide (fHBP) and at least one meningococcal capsular saccharide conjugate. In preferred embodiments, the compositions are stable and induce an immune response against strains expressing fHBP variants that are homologous to the fHBP variant in the multi-component composition and against strains expressing fHBP variants that are non-homologous to the fHBP variant in the multi-component composition.

[0127] In some embodiments, the liquid MnB divalent rLP2086 composition can be easily reconstituted with the lyophilized MenACWY-TT composition, and the combined compositions are compatible and stable. In one aspect, the disclosure relates to a composition against Neisseria meningitidis, the composition comprising: (a) a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2; and (c) a Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, the linker conjugated to a tetanus toxoid carrier protein (TT) via carbodiimide chemistry. AH (d) a meningococcal serogroup C (MenC) capsular saccharide conjugated to an ADH linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, the linker conjugated to a tetanus toxoid carrier protein (TT) via carbodiimide chemistry (MenC AH-TT conjugate), and (e) Neisseria meningitidis serovar W directly conjugated to the tetanus toxoid carrier protein (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker. 135 (MenW) capsular saccharide (MenW-TT conjugate), and (f) meningococcal serogroup Y (MenY) capsular saccharide directly conjugated to the tetanus toxoid carrier protein (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate).

[0128] In another aspect, the present disclosure relates to a composition comprising a combination of an MnB bivalent rLP2086 composition and a MenACWY-TT composition. An MnB bivalent rLP2086 composition refers to a composition comprising a single meningococcal polypeptide component that induces a broadly protective immune response effective against multiple strains of meningococcal serogroup B. Specifically, in one embodiment, the MnB bivalent rLP2086 composition comprises an MnB rLP2086 subfamily A protein (SEQ ID NO: 1) and an MnB rLP2086 subfamily B protein (SEQ ID NO: 2). In one embodiment, the composition does not comprise a fusion protein. In one embodiment, the composition does not comprise a chimeric protein. In one embodiment, the composition does not comprise a hybrid protein. In one embodiment, the composition does not further comprise a peptide fragment. In another embodiment, the composition does not further comprise a Neisserial polypeptide that is not fHBP. For example, in one embodiment, the composition does not comprise a PorA protein. In another embodiment, the composition does not comprise a NadA protein. In another embodiment, the composition does not further comprise a Neisserial heparin-binding antigen (NHBA). In another embodiment, the composition does not further comprise Neisseria outer membrane vesicles (OMVs). In a preferred embodiment, the composition does not further comprise antigens other than the first polypeptide and the second polypeptide. In a preferred embodiment, the MnB bivalent rLP2086 composition further comprises polysorbate-80. In one embodiment, the MnB bivalent rLP2086 composition further comprises a histidine buffer. In one embodiment, the MnB bivalent rLP2086 composition further comprises sodium chloride. In one embodiment, the MnB bivalent rLP2 The 086 composition further comprises aluminum phosphate. In one embodiment, the MnB divalent rLP2086 composition further comprises polysorbate-80, a histidine buffer, sodium chloride, and aluminum phosphate. Preferably, the MnB divalent rLP2086 composition is a liquid formulation, wherein the polypeptide is formulated at 120 mcg / mL per subfamily in 150 mM sodium chloride (NaCl) containing 10 mM histidine buffer, pH 6.0, 0.5 mg / mL aluminum phosphate (AlPO), and further comprises 0.018 mg polysorbate-80 per 0.5 mL dose.

[0129] A MenACWY-TT composition refers to a composition comprising purified capsular polysaccharides of Neisseria meningitidis serotypes A, C, W-135, and Y, each independently conjugated to TT at ratios (TT to polysaccharide) of about 3, about 3, about 1.5, and about 1.3, respectively. Specifically, the composition comprises (c) a meningococcal serotype A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, the linker conjugated to a tetanus toxoid carrier protein (TT) via carbodiimide chemistry. AH (d) a meningococcal serogroup C (MenC) capsular saccharide conjugated to an ADH linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, the linker conjugated to a tetanus toxoid carrier protein (TT) via carbodiimide chemistry (MenC AH -TT conjugate), and (e) Neisseria meningitidis serovar W directly conjugated to the tetanus toxoid carrier protein (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker. 135(MenW) capsular saccharide (MenW-TT conjugate), and (f) meningococcal serogroup Y (MenY) capsular saccharide directly conjugated to the tetanus toxoid carrier protein (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate). Preferably, the MenACWY-TT composition is presented as a lyophilized powder.

[0130] MenA AH -TT, MenC AH The -TT, MenW-TT, and MenY-TT conjugates are prepared by the following steps: preparation of a polysaccharide drug substance intermediate, preparation of a TT drug substance intermediate, microfluidization of the polysaccharide, derivatization of the polysaccharide (MenAAH-TT and MenCAH-TT processes only), further purification of TT, and conjugation of the individual polysaccharides with TT.

[0131] MenA AH For the -TT conjugate, MenA polysaccharide is first microfluidized to reduce molecular size and viscosity, and then activated via cyanylation with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP). The activated MenA is derivatized with adipic acid dihydrazide (ADH) to form the MenA. AH MenA AH and tetanus toxoid (TT) were coupled by carbodiimide-mediated condensation (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) coupling technique) to form MenA. AH -Tetanus toxoid conjugate (MenA AH -TT).

[0132] MenC AH For the -TT conjugate, MenC polysaccharide is first microfluidized to reduce molecular size and viscosity, and then activated via cyanylation with CDAP. The activated MenC is derivatized with adipic acid dihydrazide (ADH) to form MenC. AH MenC is formed.AH and TT were coupled by carbodiimide-mediated condensation (EDAC coupling technique) to give MenC AH -Tetanus toxoid (MenC AH -TT).

[0133] For the MenW-TT conjugate, MenW polysaccharide is first microfluidized to reduce molecular size and viscosity, and then activated via cyanylation with CDAP. The activated MenW is then directly coupled with TT to form MenW-tetanus toxoid (MenW-TT).

[0134] For the MenY-TT conjugate, MenY polysaccharide is first microfluidized to reduce molecular size and viscosity, and then activated via cyanylation with CDAP. The activated MenY is then directly coupled with TT to form MenY-tetanus toxoid (MenY-TT).

[0135] In another aspect, polypeptide antigens derived from up to two meningococcal serogroup B strains induce a broadly protective immune response effective against multiple strains of meningococcal serogroup B. Thus, in one embodiment, the composition does not further comprise a polypeptide not derived from Neisseria meningitidis serogroup B fHBP subfamily A strain M98250771 and / or Neisseria meningitidis serogroup B fHBP subfamily B strain CDC1573.

[0136] In one embodiment, the composition does not further comprise a polypeptide having less than 100% sequence identity to SEQ ID NO: 1. In another embodiment, the composition does not further comprise a polypeptide having less than 100% sequence identity to SEQ ID NO: 2. For example, the composition does not further comprise a polypeptide having less than 100% sequence identity to the full length of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0137] In one embodiment, the composition further comprises polysorbate-80, aluminum, histidine, and sodium chloride. In one embodiment, the composition comprises about 60 μg of a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, about 60 μg of a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, polysorbate-80 at a molar ratio of 2.8 to each polypeptide, 0.5 mg aluminum / mL as aluminum phosphate, 10 mM histidine, and 150 mM sodium chloride, and the composition preferably has a total volume of about 0.5 mL.

[0138] In another aspect, a composition comprises about 120 μg / mL of a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, about 120 μg / mL of a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, polysorbate-80 at a molar ratio of 2.8 to each polypeptide, 0.5 mg aluminum / mL as aluminum phosphate, 10 mM histidine, and 150 mM sodium chloride.

[0139] In a further aspect, a composition comprises: a) 60 μg of a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; b) 60 μg of a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2; c) 18 μg of polysorbate-80; d) 250 μg of aluminum; e) 780 μg of histidine; and f) 4380 μg of sodium chloride.

[0140] In an exemplary embodiment, the composition comprises about 60 μg of a first lipidated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1, about 60 μg of a second lipidated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a 2.8 molar ratio of polysorbate-80 to the first lipidated polypeptide and to the second lipidated polypeptide, 0.5 mg / mL aluminum phosphate, 10 mM histidine, and 150 mM sodium chloride, and the composition has a total volume of about 0.5 mL. In an exemplary embodiment, the composition is a suspension in a sterile isotonic buffer. In an exemplary embodiment, the composition has a pH of 6.0. In an exemplary embodiment, the first polypeptide and the second polypeptide are adsorbed to aluminum.

[0141] In one embodiment, the composition comprises a MenA having an average TT / polysaccharide ratio of 3. AH -TT conjugate and MenC with an average TT / polysaccharide ratio of 3 AH In a preferred embodiment, the composition comprises a MenA polysaccharide having 5 mcg of MenA polysaccharide and about 15 mcg of TT, a MenW-TT conjugate having an average TT / polysaccharide ratio of 1.5, and a MenY-TT conjugate having an average TT / polysaccharide ratio of 1.3. AH -TT conjugate and MenC with 5 mcg of MenC polysaccharide and approximately 15 mcg of TT AH a MenW-TT conjugate having 5 mcg of MenW polysaccharide and about 7.5 mcg of TT, and a MenY-TT conjugate having 5 mcg of MenY polysaccharide and about 6.5 mcg of TT. The composition may further include Tris-HCl, sucrose, and sodium chloride.

[0142] In another embodiment, the composition comprises MenA polysaccharides, MenC polysaccharides, MenW polysaccharides, and MenY polysaccharides, and TT carrier protein. AH -TT conjugate, MenC AHThe compositions include a MenA-TT conjugate, a MenW-TT conjugate, and a MenY-TT conjugate. The compositions may further include sucrose and trometanol. For example, in one embodiment, the composition includes 10 μg / mL MenA polysaccharide, 10 μg / mL MenC polysaccharide, 10 μg / mL MenW polysaccharide, and 10 μg / mL MenY polysaccharide, 88 μg / mL TT carrier protein, 164 mM sucrose, and 1.6 mM trometanol.

[0143] In one embodiment, the composition has a total volume of about 0.5 mL. In one embodiment, the first dose of the composition has a total volume of about 0.5 mL. "First dose" refers to the dose of the composition administered on day 0. "Second dose" or "third dose" refers to the dose of the composition administered subsequent to the first dose, which may or may not be the same amount as the first dose.

[0144] In one aspect, the present disclosure relates to a liquid immunogenic composition resulting from a lyophilized MenACWY-TT composition that has been reconstituted with a liquid MnB bivalent rLP2086 composition. Reconstitution refers to restoring a dry, lyophilized composition to a liquid form by adding a liquid diluent. In a preferred embodiment, the liquid MnB bivalent rLP2086 composition is not administered concomitantly, coadministered, or simultaneously with a lyophilized MenACWY-TT composition, and the lyophilized MenACWY-TT composition is reconstituted with a liquid composition that is not a liquid MnB bivalent rLP2086 composition. For example, in a preferred embodiment, the lyophilized MenACWY-TT composition is not reconstituted with an aqueous diluent consisting of sodium chloride and water, and subsequently not administered concomitantly, coadministered, or simultaneously with a liquid MnB bivalent rLP2086 composition.

[0145] Instead, in a preferred embodiment, the lyophilized MenACWY-TT composition is administered together with the MnB bivalent rLP2086 composition in a single dose to a human. The resulting single dose (e.g., the MenABCWY composition) can result from mixing the MnB bivalent rLP2086 composition from a first container with the lyophilized MenACWY-TT composition from a second container. Alternatively, the single dose of the MenABCWY composition can result from one (single) container containing the MnB bivalent rLP2086 composition and the lyophilized MenACWY-TT composition. Delivery devices for vaccines or immunogenic compositions are known in the art. In one embodiment, the MenABCWY composition is administered in combination with any one of ibuprofen, paracetamol, and amoxicillin.

[0146] The composition is immunogenic after administration of the first dose to a human, hi one embodiment, the first dose is in a total volume of about 0.5 mL. The composition induces a serum immunoglobulin bactericidal titer in a human after receiving the first dose that is at least one-fold higher, preferably at least two-fold higher, than the serum immunoglobulin bactericidal titer in the human before receiving the first dose, as measured in a serum bactericidal assay (hSBA) using human complement under identical conditions.

[0147] The bactericidal titer or bactericidal immune response is against meningococcal serotype B. In a preferred embodiment, the bactericidal titer or bactericidal immune response is against meningococcal serotype B fHBP subfamily A strains and against meningococcal serotype B fHBP subfamily B strains. Most preferably, the bactericidal titer or bactericidal immune response is against at least meningococcal serotype B, fHBP subfamily B, strain B01.

[0148] In one aspect, the composition induces a bactericidal titer of serum immunoglobulin in a human after receiving a dose of the composition that is at least 1-fold higher, such as at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 16-fold higher, than the bactericidal titer of serum immunoglobulin in a human before receiving said dose, when measured in a serum bactericidal assay using human complement under the same conditions.

[0149] In one embodiment, the composition is an immunogenic composition. In one embodiment, the composition is an immunogenic composition for humans. In another embodiment, the composition is a vaccine. A "vaccine" refers to a composition comprising an antigen, which contains at least one epitope that induces an immune response specific to that antigen. The vaccine may be administered directly into a subject by subcutaneous, oral, oronasal, or intranasal routes of administration. Preferably, the vaccine is administered intramuscularly. In one embodiment, the composition is a human vaccine. In one embodiment, the composition is an immunogenic composition against Neisseria meningitidis.

[0150] In one embodiment, the composition is a liquid composition. In a preferred embodiment, the composition is a liquid suspension composition. In another preferred embodiment, the composition is not lyophilized. stability The terms "stable" and "stability" refer to the ability of an antigen to remain immunogenic over a period of time. Stability can be measured by potency over time. The terms "stable" and "stability" further refer to the physical, chemical, and conformational stability of an immunogenic composition. Instability of a protein composition can be caused by chemical degradation or aggregation of protein molecules to form higher-order polymers, dissociation of heterodimers into monomers, deglycosylation, modified glycosylation, or any other structural modification that reduces at least one biological activity of a protein composition encompassed by the present disclosure. Stability can be assessed 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 differential scanning calorimetry (DSC) characteristics. Other methods for assessing stability are known in the art and can also be used in accordance with the present disclosure.

[0151] In some embodiments, the antigen in the stable formulation of the present disclosure may maintain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% potency compared to a reference standard 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, 54 months, or 60 months. In some embodiments, the antigen in the stable formulation of the present disclosure may maintain at least 50% potency compared to a reference standard for at least 1 year, 2 years, 3 years, 4 years, or 5 years. The terms "stable" and "stability" also refer to the ability of an antigen to maintain epitope or immune reactivity over a period of time. For example, the antigen in the stable formulation of the present disclosure may be The antigen may maintain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of its epitope or immunoreactivity 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, 54 months, or 60 months, compared to a reference standard. In some embodiments, stability is measured with respect to environmental conditions. Non-limiting examples of environmental conditions include light, temperature, freeze-thaw cycles, agitation, and pH. One of skill in the art would be able to determine the presence of antigen epitopes or immunoreactivity using the methods disclosed herein or other methods known in the art. In some embodiments, the stability of the antigen is measured from the date of its formulation. In some embodiments, the stability of an antigen is measured from the date of a change in its storage conditions, non-limiting examples of which include a change from frozen to refrigerated, from frozen to room temperature, from refrigerated to room temperature, from refrigerated to frozen, from room temperature to frozen, from room temperature to refrigerated, from light to dark, or the introduction of agitation.

[0152] In one embodiment, the terms "stable" and "stability" include the ability of an antigen to bind to aluminum. For example, a stable formulation of the present disclosure comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the protein in the formulation bound to aluminum (e.g., aluminum phosphate) for at least 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 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, 54 months, or 60 months, compared to a reference standard. See, e.g., Example 13. In preferred embodiments, at least 90%, more preferably at least 95%, and most preferably at least 99% of all subfamily A rLP2086 polypeptides (e.g., polypeptides comprising the amino acid sequence set forth in SEQ ID NO:1) are aluminum-bound in the composition. In preferred embodiments, at least 90%, more preferably at least 95%, and most preferably at least 99% of all subfamily B rLP2086 polypeptides (e.g., polypeptides comprising the amino acid sequence set forth in SEQ ID NO:2) are aluminum-bound in the composition.

[0153] Determination of Aluminum Binding. Compositions containing aluminum and at least one protein antigen were centrifuged to pellet the aluminum. Centrifugation of aluminum-absorbed proteins is known in the art. See, for example, Egan et al., Vaccine, 27(24):3175-3180 (2009). Aluminum-binding proteins were also pelleted, while non-aluminum-binding proteins remained in the supernatant. Total protein in the supernatant and pellet was determined by Lowry assay. The percentage of bound protein was calculated by dividing the total protein in the supernatant by the total protein added to the composition and multiplying by 100%. Similarly, the percentage of unbound protein was calculated by dividing the total protein in the supernatant by the total protein added to the composition and multiplying by 100%. For compositions containing both subfamily A and subfamily B antigens, the concentrations of individual subfamily A and B proteins in the supernatant were determined by ion exchange chromatography. Separation and elution of subfamily A and B proteins were performed using a strong anion column and a high salt eluent. Both subfamily A and B proteins were detected and quantified using a fluorescence detector set at excitation = 280 run and emission = 310 run. Subfamily A and subfamily B proteins eluted at distinct retention times and were quantified using a calibration curve constructed against the rLP2086 protein reference material. The percentage of unbound protein was calculated by dividing the total protein in the supernatant by the total protein added to the composition and multiplying by 100%. The percentage of bound protein was calculated by subtracting the percentage of unbound protein from 100%. Polysorbate-80 Polysorbate 80 (PS-80) is a nonionic surfactant. Accelerated stability studies using an in vitro monoclonal antibody-based potency assay demonstrated that subfamily B proteins were unstable at higher molar ratios of PS-80 to MnB rLP2086 protein in the final formulation. Further experiments with various ratios of PS-80 demonstrated that the optimal molar ratio of PS-80 to MnB rLP2086 protein for retaining potency was approximately 2.8 ± 1.4.

[0154] The concentration of PS-80 in the composition depends on the molar ratio of PS-80 to polypeptide. In one embodiment, the composition comprises a molar ratio of PS-80 to the first polypeptide and to the second polypeptide of 2.8±1.4. In one embodiment, the composition comprises a molar ratio of PS-80 to the first polypeptide and to the second polypeptide of 2.8±1.1. In one embodiment, the composition comprises a molar ratio of PS-80 to the polypeptide of at least 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3. In one embodiment, the composition comprises a molar ratio of PS-80 to the polypeptide of at most 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, or 2.9. Any minimum value may be combined with any maximum value described herein to define a range. Preferably, the composition comprises a molar ratio of PS-80 to polypeptide of 2.8.

[0155] The molar ratio of PS-80 to polypeptide is determined by calculation from the measured concentration of PS-80 and the measured concentration of total polypeptide, both values ​​expressed in moles. For example, the molar ratio of PS-80 to protein is determined by calculation from the measured concentration of PS-80 (e.g., by reverse-phase high-pressure liquid chromatography (RP-HPLC)) to the measured concentration of total protein (e.g., by ion-exchange high-pressure liquid chromatography (IEX-HPLC)), both values ​​expressed in moles, in the final drug substance.

[0156] RP-HPLC is used to quantify the concentration of polysorbate 80 in vaccine formulations. The detergent concentration is determined by saponification of the fatty acid moiety. Polysorbate 80 is converted to free oleic acid by alkaline hydrolysis at 40°C. Samples are separated by RP-HPLC using a C18 column and quantified using a UV detector at a wavelength of 200 nm.

[0157] The first and second polypeptides are resolved by anion exchange HPLC. The rLP2086 (fHBP) subfamily A and B proteins are eluted at distinct retention times and quantified using calibration curves generated against the respective rLP2086 protein reference materials.

[0158] The term "molar ratio" and an explanation of immunogenic compositions comprising fHBP and PS-80 are further disclosed in WO2012025873 and U.S. Patent Publication No. US2013 / 0171194, each of which is incorporated by reference in its entirety.

[0159] As used herein, the term "molar ratio" refers to the ratio of the moles of two different elements in a composition.In some embodiments, the molar ratio is the ratio of the moles of detergent to the moles of polypeptide.In some embodiments, the molar ratio is the ratio of the moles of PS-80 to the moles of protein.In one embodiment, based on the concentration of protein and polysorbate 80, the molar ratio can be calculated using the following equation:

[0160]

number

[0161] In one embodiment, the composition comprises a molar ratio of PS-80 to MnB rLP2086 protein of 1.4 to 4.2 to maintain potency. In one embodiment, the composition comprises at least 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, or 2.8. In one embodiment, the composition comprises at most 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, or 2.8. Any minimum value may be combined with any maximum value described herein to define a range.

[0162] In one embodiment, the composition comprises about 0.0015, 0.0017, 0.0019, 0.0021, 0.0023, 0.0025, 0.0027, 0.0029, 0.0031, 0.0033, 0.0035, 0.0037, 0.0039, 0.0041, 0.0043, 0.0045, 0.0047, 0.0049, 0.0051 mg / mL of PS-80. Preferably, the composition comprises about 0.0035 mg / mL of PS-80.

[0163] In another embodiment, the composition contains at least 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, or 25 μg of PS-80. In another embodiment, the composition contains at most 30 μg, 29 μg, 28 μg, 27 μg, 26 μg, 25 μg, 24 μg, 23 μg, 22 μg, 21 μg, 20 μg, 19 μg, or 18 μg of PS-80. Any minimum value may be combined with any maximum value described herein to define a range. In a preferred embodiment, the composition contains at least 10 μg and at most 20 μg of PS-80. In a most preferred embodiment, the composition contains about 18 μg of PS-80.

[0164] In another embodiment, the composition comprises a PS-80 concentration in the range of 0.0005% to 1%. For example, the PS-80 concentration in the composition can be at least 0.0005%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or 1.1% PS-80. In one embodiment, the PS-80 concentration in the composition can be up to 2.0%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, or 0.7% PS-80. In a preferred embodiment, the composition contains about 0.07% PS-80. Any minimum value can be combined with any maximum value described herein to define a range.

[0165] In some embodiments, a composition comprising a combination of a first composition and a second composition may have a different molar ratio of polysorbate-80 to MnB rLP2086 polypeptide compared to the molar ratio of polysorbate-80 to MnB rLP2086 polypeptide in the first composition. In some embodiments, no additional surfactant is required in the combined composition to maintain the solubility and stability of the MnB rLP2086 polypeptide in the combined composition. Thus, in one embodiment, the kit does not contain more than 0.02 mg of polysorbate-80. aluminum The composition includes aluminum as aluminum phosphate. AlPO4 is added as a stabilizer to provide enhanced manufacturability and stability. The process is described in U.S. Patent Publication US2009 / 0016946, which is incorporated by reference in its entirety. In one embodiment, the composition does not further comprise a polyvalent cation other than aluminum. In one embodiment, the composition does not further comprise Al(OH) or Al(SO).

[0166] In one embodiment, the composition comprises at least 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, or 250 μg of aluminum. In one embodiment, the composition contains at most 500 μg, 490 μg, 480 μg, 470 μg, 460 μg, 450 μg, 440 μg, 430 μg, 420 μg, 410 μg, 400 μg, 390 μg, 380 μg, 370 μg, 360 μg, 350 μg, 340 μg, 330 μg, 320 μg, 310 μg, 300 μg, 290 μg, 280 μg, 270 μg, 260 μg, or 250 μg of aluminum. Any minimum value may be combined with any maximum value described herein to define a range. In a most preferred embodiment, the composition contains 250 μg of aluminum.

[0167] In one embodiment, the composition comprises at least 0.005 mg / ml, 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.10 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, or 0.5 mg / ml of aluminum phosphate. In one embodiment, the composition comprises at most 2.0 mg / ml, 1.9 mg / ml, 1.8 mg / ml, 1.7 mg / ml, 1.6 mg / ml, 1.5 mg / ml, 1.4 mg / ml, 1.3 mg / ml, 1.2 mg / ml, 1.1 mg / ml, 1.0 mg / ml, 0.9 mg / ml, 0.8 mg / ml, or 0.7 mg / ml of PS-80. In a preferred embodiment, the composition contains about 0.07 mg / ml PS-80. Any minimum value may be combined with any maximum value described herein to define a range. In a preferred embodiment, the composition contains 0.5 mg / ml aluminum phosphate. In a most preferred embodiment, the composition contains 0.5 mg aluminum / ml as aluminum phosphate (AlPO). This concentration maintains aluminum binding (at least 90% binding or greater) to subfamily A and B proteins.

[0168] In some embodiments, the combination of the first and second compositions alters the percentage of MnB rLP2086 polypeptide bound to aluminum compared to the percentage of MnB rLP2086 polypeptide bound to aluminum in the first composition. In some embodiments, the combination of the first and second compositions maintains at least 90% binding of total MnB rLP2086 polypeptide to aluminum. Thus, in one embodiment, the percentage of total MnB rLP2086 polypeptide to aluminum in the combined composition is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Preferably, the percentage of total MnB rLP2086 polypeptide to aluminum in the combined composition is at least 90%, more preferably at least 95%, and most preferably at least 100%.

[0169] In another embodiment, the concentration of aluminum-bound polypeptide in the immunogenic composition is not decreased after 24 hours compared to the concentration of aluminum-bound polypeptide in the liquid composition before reconstitution of the lyophilized composition. AH The concentration of the MenA-TT conjugate in the lyophilized composition AH In one embodiment, the concentration is not decreased after 24 hours compared to the concentration of the -TT conjugate. The concentration is reduced by up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% after 24 hours compared to the respective concentration in the liquid composition before dissolution.

[0170] In another embodiment, the immunogenic composition comprises MenC. AH The concentration of the MenC-TT conjugate in the lyophilized composition AHIn another embodiment, the concentration of the MenW-TT conjugate in the immunogenic composition is not decreased after 24 hours compared to the concentration of the MenW-TT conjugate in the lyophilized composition. In another embodiment, the concentration of the MenY-TT conjugate in the immunogenic composition is not decreased after 24 hours compared to the concentration of the MenY-TT conjugate in the lyophilized composition. In one embodiment, the concentration is reduced by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% after 24 hours compared to the respective concentrations in the lyophilized composition before reconstitution. excipients In one embodiment, the composition comprises histidine. In one embodiment, the composition comprises at least 650 μg, 660 μg, 670 μg, 680 μg, 690 μg, 700 μg, 710 μg, 720 μg, 730 μg, 740 μg, 750 μg, 760 μg, 770 μg, 780 μg, 790 μg, 800 μg, 810 μg, 820 μg, 830 μg, 840 μg, or 850 μg of histidine. In one embodiment, the composition contains up to 1560 μg, 1500 μg, 1400 μg, 1300 μg, 1200 μg, 1100 μg, 1000 μg, 950 μg, 900 μg, 890 μg, 880 μg, 870 μg, 860 μg, 850 μg, 840 μg, 830 μg, 820 μg, 810 μg, 800 μg, 790 μg, or 780 μg of histidine. Any minimum value can be combined with any maximum value described herein to define a range. Preferably, the composition contains 780 μg of histidine.

[0171] In one embodiment, the composition comprises a Tris, phosphate, or succinate buffer. In a preferred embodiment, the composition does not comprise a Tris buffer. In a preferred embodiment, the composition does not comprise a phosphate buffer. In a preferred embodiment, the composition does not comprise a succinate buffer. In a preferred embodiment, the composition comprises a histidine buffer.

[0172] In one embodiment, the composition comprises sodium chloride. The sodium chloride concentration in the MenABCWY composition can vary from 160.5 to 161.1 mM. In one embodiment, the pH of the composition is 5.5 to 7.5. In a preferred embodiment, the pH of the composition is 5.8 to 7.0, and most preferably 5.8 to 6.0. In one embodiment, the pH of the composition is up to 6.1. In one embodiment, the pH of the composition is 5.8. kit A further aspect of the present disclosure is a kit for administering a dose of a composition for inducing bactericidal antibodies against meningococcus in a mammal.

[0173] In one aspect, the kit comprises a first composition comprising the first polypeptide described above and the second polypeptide described above. In a preferred embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In another preferred embodiment, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. The kit comprises a MenA AH -TT conjugate, MenC AH In one embodiment, the kit further comprises a second composition comprising a MenY-TT conjugate, a MenW-TT conjugate, and a MenY-TT conjugate. In one embodiment, the kit comprises at least two containers, a first container comprising the first composition and a second container comprising the second composition.

[0174] In one embodiment, the kit comprises a liquid first composition and a lyophilized second composition. Preferably, the kit comprises a liquid MnB bivalent rLP2086 composition and a lyophilized MenA Contains CWY-TT composition.

[0175] In some embodiments, the composition comprises a combination of a first composition and a second composition, which alters the molar ratio of polysorbate-80 to MnB rLP2086 polypeptide in the first composition. In some embodiments, no additional surfactant is required in the combined composition to maintain the solubility and stability of the MnB rLP2086 polypeptide in the combined composition. Thus, in one embodiment, the kit does not contain more than 0.02 mg of polysorbate-80.

[0176] In one embodiment of the disclosure, the kit does not further include any one of the following commercially available immunogenic compositions: MENACTRA®, MENVEO®, ADACEL®, HAVRIX®, GARDASIL®, REPEVAX, or any combination thereof. For example, preferably, the kit does not further include a meningococcal A, C, Y, and W-135 polysaccharide conjugate (MCV4) composition and the carrier protein is diphtheria toxoid. In one embodiment, the kit does not further include a meningococcal A, C, Y, and W-135 polysaccharide conjugate (MCV4) composition and the carrier protein is CRM 197 In one embodiment, the kit does not further comprise the NIMENRIX vaccine, and the NIMENRIX comprises a diluent consisting of sodium chloride and water. Bactericidal activity The disease incidence of MnB is approximately 1 in 100,000, meaning that a very large number of subjects (400,000 to over 6 million) is required to support a statistically significant assessment of efficacy. Therefore, the human complement-based serum bactericidal assay (hSBA), a surrogate for protection and vaccine efficacy, is used to assess immunogenicity in clinical trials.

[0177] Pfizer has been building a large collection of MnB strains (N = at least 1263), including isolates that cause IMD, from 2000 to 2006. MnB isolates were systematically collected from the US Centers for Disease Control and Prevention (CDC) and health and reference laboratories in European countries.

[0178] In one embodiment, the immune response induced by administering the composition to a human is determined using a human complement-based serum bactericidal assay (hSBA) against four meningococcal serogroup B (MnB) strains. The MnB strains used in the hSBA were selected from a strain pool, which represents a systematically curated collection of clinically relevant meningococcal strains.

[0179] The high percentage of hSBA responses against all test strains, particularly strains expressing lipoprotein 2086 variants with sequences non-homologous to both the first and second polypeptides, suggests that the composition is a broadly protective vaccine and is sufficient to confer high seroprotection against meningococcal strains expressing rLP2086 (FHBP) from at least serogroup B, including additional serogroups such as serogroup X. Subfamily A strains In one embodiment, the hSBA strain is a meningococcal strain expressing the LP2086 (fHBP) subfamily A protein. In one embodiment, the hSBA strain is a LP2086 (fHBP) subfamily A strain expressing a lipoprotein 2086 variant that is heterologous to meningococcal strains expressing A05. For example, in one embodiment, the hSBA strain is a LP2086 (fHBP) subfamily A strain expressing a lipoprotein 2086 variant that is heterologous to strain M98250771.

[0180] In one embodiment, the hSBA strain is a meningococcal strain expressing fHBP A10. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086(fHBP)A22. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086(fHBP)A56. In a further embodiment, the hSBA strain is an LP2086(fHBP)A22 and LP2086(fHBP)A56 strain. In another embodiment, the hSBA strain is a meningococcal strain expressing LP2086 A04. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 A05. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 A12. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 A22. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 A12. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 A04. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 A19. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 A07. In a further embodiment, the hSBA strain comprises any one of A22, A12, A19, A05, and A07 expressing strains. In one embodiment, the hSBA strain comprises any one of A06, A15, and A29 expressing strains.

[0181] In one embodiment, the immune response is directed against a meningococcal serogroup B strain heterologous to the A05-expressing meningococcal strain. bactericidal against fHPB subfamily A strains. In one embodiment, the immune response is against meningococcal serogroup B A22 strain. In one embodiment, the immune response is against meningococcal serogroup B In one embodiment, the immune response is against meningococcal serogroup B subfamily A strain A56. In one embodiment, the immune response is against meningococcal serogroup B A06 strain. In one embodiment, the immune response is against meningococcal serogroup B A15 strain. In one embodiment, the immune response is against meningococcal serogroup B A29 strain. In one embodiment, the immune response is against meningococcal serogroup B A62 strain. In one embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily A strains heterologous to meningococcal strain M98250771.

[0182] In one aspect, the immune response is bactericidal against a meningococcal serogroup B subfamily A strain expressing a factor H binding protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. In another embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily A strains expressing a factor H binding protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the factor H binding protein expressed by meningococcal strain M98250771. In a preferred embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily A strains expressing a factor H binding protein comprising an amino acid sequence having at least 80%, more preferably at least 84%, identity to the factor H binding protein expressed by meningococcal strain M98250771.

[0183] In another aspect, the immune response is bactericidal against a meningococcal serogroup B subfamily A strain expressing a factor H binding protein comprising an amino acid sequence having at most 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. In another embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily A strains expressing a factor H binding protein comprising an amino acid sequence having at most 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the factor H binding protein expressed by meningococcal strain M98250771. In a preferred embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily A strains expressing a factor H binding protein comprising an amino acid sequence having at most 85%, more preferably at most 99%, identity to the factor H binding protein expressed by meningococcal strain M98250771. It is bactericidal against Serotype B subfamily A strains. Any minimum value may be combined with any maximum value described herein to define a range.

[0184] In one embodiment, the immune response elicited by the composition is bactericidal not only against meningococcal serogroup B fHPB subfamily A strains, but also against meningococcal strains that express fHBP subfamily A polypeptides that are not serogroup B. For example, in a preferred embodiment, the immune response elicited by the composition is bactericidal against meningococcal serogroup B subfamily A strains and against meningococcal serogroup C strains that express fHBP subfamily A polypeptides that are non-homologous to fHBP A05. For example, in one embodiment, the immune response is against meningococcal serogroup C strains that express fHBP A10. In another embodiment, the immune response is against meningococcal serogroup W strains that express fHBP A19. In one embodiment, the immune response is bactericidal against meningococcal strains that express fHBP subfamily A polypeptides that are non-homologous to meningococcal strain M98250771. Subfamily B strains In one embodiment, the hSBA strain is an LP2086(fHBP) subfamily B strain. In one embodiment, the hSBA strain is an LP2086(fHBP) subfamily B strain that expresses a lipoprotein 2086 variant that is heterologous to meningococcal strains that express B01. For example, in one embodiment, the hSBA strain is an LP2086(fHBP) subfamily B strain that expresses a lipoprotein 2086 variant that is heterologous to strain CDC1127. In a preferred embodiment, the hSBA strain is an LP2086(fHBP) subfamily B strain that expresses a lipoprotein 2086 variant that is heterologous to strain CDC1573.

[0185] In one aspect, the immune response is directed against a meningococcal serogroup B strain that is heterologous to the meningococcal strain expressing B01. bactericidal against fHPB subfamily B strains. In one embodiment, the immune response is against meningococcal serogroup B B24 strain. In one embodiment, the immune response is against meningococcal serogroup B In one embodiment, the immune response is against meningococcal serogroup B strain B44. In one embodiment, the immune response is against meningococcal serogroup B strain B16. In one embodiment, the immune response is against meningococcal serogroup B strain B03. In one embodiment, the immune response is against meningococcal serogroup B strain B09. In one embodiment, the immune response is against meningococcal serogroup B strain B15. In one embodiment, the immune response is against meningococcal serogroup B strain B153. In one embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily B strains that are heterologous to meningococcal strain CDC1573.

[0186] In one aspect, the immune response is bactericidal against a meningococcal serogroup B subfamily B strain expressing a factor H binding protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the second polypeptide. In another embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily B strains expressing a factor H binding protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the factor H binding protein expressed by meningococcal strain CDC1573. In a preferred embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily B strains expressing a factor H binding protein comprising an amino acid sequence having at least 80% identity, more preferably at least 87% identity, to the factor H binding protein expressed by meningococcal strain CDC1573. In another preferred embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily B strains that express a factor H binding protein that comprises an amino acid sequence that has 100% identity to the factor H binding protein expressed by meningococcal strain CDC1573.

[0187] In another embodiment, the immune response is bactericidal against a meningococcal serogroup B subfamily B strain expressing a factor H binding protein comprising an amino acid sequence having at most 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the second polypeptide. In another embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily B strains expressing a factor H binding protein comprising an amino acid sequence having at most 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the factor H binding protein expressed by meningococcal strain CDC1573. In a preferred embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily B strains expressing a factor H binding protein comprising an amino acid sequence having at most 88% identity, more preferably at least 99% identity, to the factor H binding protein expressed by meningococcal strain CDC1573. Any minimum value may be combined with any maximum value described herein to define a range.

[0188] In one embodiment, the hSBA strain is strain LP2086(fHBP)B24. In another embodiment, the hSBA strain is strain LP2086(fHBP)B44. In a further embodiment, the hSBA strain comprises strains LP2086(fHBP)B24 and LP2086(fHBP)B44. In one embodiment, the hSBA strain comprises strains LP2086(fHBP)A22, LP2086(fHBP)A56, LP2086(fHBP)B24, and LP2086(fHBP)B44. In one embodiment, the hSBA strain comprises B15. In one embodiment, the hSBA strain comprises B153. In another embodiment, the hSBA strain is strain LP2086 B16. In one embodiment, the hSBA strain is strain LP2086 B03. In one embodiment, the hSBA strain is LP2086 B09 strain. In a further embodiment, the hSBA strain comprises B24, B16, B44, B03, and B09, or any combination thereof. In another embodiment, the hSBA strain comprises B24, B16, B44, A22, B03, B09, A12, A19, A05, and A07, or any combination thereof. In another embodiment, the hSBA strain comprises A06, A07, A12, A15, A19, A29, B03, B09, B15, and B16, or any combination thereof.

[0189] In one embodiment, the method induces an immune response against Neisseria meningitidis serogroup B fHPB subfamily A strains and Neisseria meningitidis serogroup B fHPB subfamily B strains. Preferably, the immune response is bactericidal against Neisseria meningitidis serogroup B fHPB subfamily A strains and Neisseria meningitidis serogroup B fHPB subfamily B strains.

[0190] In one embodiment, the immune response elicited by the composition is bactericidal not only against meningococcal serogroup B fHPB subfamily B strains, but also against meningococcal strains expressing fHBP subfamily B polypeptides that are not serotype B. For example, in a preferred embodiment, the immune response elicited by the composition is bactericidal against meningococcal serogroup B subfamily B strains and against meningococcal serogroup Y strains expressing fHBP subfamily B polypeptides that are non-homologous to fHBP B01. For example, in one embodiment, the immune response is against meningococcal serogroup A strains expressing fHBP B16. In another embodiment, the immune response is against meningococcal serogroup Y strains expressing fHBP B47. In another embodiment, the immune response is against meningococcal serogroup X strains expressing fHBP B49. In one embodiment, the immune response is bactericidal against meningococcal strains heterologous to meningococcal serogroup B strain CDC1573 that express fHBP subfamily B polypeptides.

[0191] In one aspect, the present invention relates to the use of a composition comprising a first lipidated polypeptide variant of Neisseria meningitidis serogroup B factor H binding protein (fHBP) and a second lipidated polypeptide variant of Neisseria meningitidis serogroup B fHBP. and eliciting a bactericidal immune response against at least one meningococcal serogroup B strain expressing a polypeptide selected from the group consisting of A42, A63, A76, B05, B07, B08, B13, B52, and B107. For example, in one aspect, the invention relates to the use of a composition comprising a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2.

[0192] In one embodiment, the hSBA strain is a meningococcal strain expressing fHBP B05. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086(fHBP)B07. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086(fHBP)B08. In another embodiment, the hSBA strain is a meningococcal strain expressing LP2086 B13. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 B52. In one embodiment, the hSBA strain is a meningococcal strain expressing LP2086 B107. In a further embodiment, the hSBA strain comprises any one strain selected from the group consisting of B05, B07, B08, B13, B52, and B107. In a further embodiment, the hSBA strain comprises any one strain selected from the group consisting of B05, B07, B08, B13, B52, B107, B01, B24, B44, B16, B03, B09, B15, and B153.

[0193] In one aspect, the immune response is bactericidal against meningococcal serogroup B subfamily B strains that are heterologous to meningococcal strains expressing B01. In one embodiment, the immune response is against a meningococcal serogroup B strain B05. In one embodiment, the immune response is against a meningococcal serogroup B strain B07. In one embodiment, the immune response is against a meningococcal serogroup B strain B08. In one embodiment, the immune response is against a meningococcal serogroup B strain B13. In one embodiment, the immune response is against a meningococcal serogroup B strain B52. In one embodiment, the immune response is against a meningococcal serogroup B strain B107. In one embodiment, the immune response is against a meningococcal serogroup B strain B24. In one embodiment, the immune response is against a meningococcal serogroup B strain B44. In one embodiment, the immune response is against a meningococcal serogroup B strain B16. In one embodiment, the immune response is against a meningococcal serogroup B strain B03. In one embodiment, the immune response is against a meningococcal serogroup B strain B09. In one embodiment, the immune response is against meningococcal serogroup B strain B15. In one embodiment, the immune response is against meningococcal serogroup B strain B153. In one embodiment, the immune response is bactericidal against meningococcal serogroup B subfamily B strains that are heterologous to meningococcal strain CDC1573.

[0194] In one embodiment, the immune response is against a meningococcal serogroup B strain selected from the group consisting of A02, A28, A42, A63, and A76. In one embodiment, the immune response is against a meningococcal serogroup B strain selected from the group consisting of B05, B07, B08, B13, B52, B107, B01, B24, B44, B16, B03, B09, B15, and B153, and any combination thereof.

[0195] In one embodiment, the hSBA strains include B05, B07, B08, B13, B52, and B107, and any combination thereof. In a further embodiment, the hSBA strains include B05, B07, B08, B13, B52, and B107, B24, B16, B44, B03, and B09, and any combination thereof. In one embodiment, the hSBA strains include A02, A28, A42, A63, A76, B05, B07, B08, B13, B52, and B107, and any combination thereof. In another embodiment, the hSBA strains further include A06, A07, A12, A15, A19, A29, B03, B09, B15, and B16, or any combination thereof. In another embodiment, the hSBA strains include A02, A28, A42, A63, A76, B05, B07, B08, B13, B52, and B107, A06, A07, A12, A15, A19, A29, B03, B09, B15, and B16, and any combination thereof.

[0196] In one aspect, the method induces an immune response against Neisseria meningitidis serogroup B subfamily A and Neisseria meningitidis serogroup B subfamily B strains. Preferably, the immune response is bactericidal against Neisseria meningitidis serogroup B subfamily A and Neisseria meningitidis serogroup B subfamily B strains. In one aspect, the method induces an immune response against Neisseria meningitidis serogroup B strains selected from the group consisting of A02, A28, A42, A63, A76, B05, B07, B08, B13, B52, and B107, and any combination thereof. In one aspect, the method induces an immune response against a meningococcal serogroup B strain selected from the group consisting of A02, A28, A42, A63, A76, B05, B07, B08, B13, B52, and B107, A06, A07, A12, A15, A19, A29, B03, B09, B15, and B16, and any combination thereof. Titer In one embodiment, the composition induces an increase in bactericidal titer in a human compared to the bactericidal titer in the human before administering a dose of the composition, when measured under the same conditions in an hSBA. In one embodiment, the increase in bactericidal titer is compared to the bactericidal titer in the human before administering a first dose of the composition, when measured under the same conditions in an hSBA. In one embodiment, an increase in titer is observed after a second dose of the composition compared to the bactericidal titer in the human before administering a second dose of the composition, when measured under the same conditions in an hSBA. In another embodiment, an increase in bactericidal titer is observed after a third dose of the composition compared to the bactericidal titer in the human before administering a third dose of the composition, when measured under the same conditions in an hSBA.

[0197] In one embodiment, the composition induces a bactericidal titer in a human after administration of the dose, when measured under the same conditions in an hSBA, that is at least 1-fold greater than the bactericidal titer in the human before administration of the dose. For example, the bactericidal titer may be at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 16-fold greater in a human after receiving a dose of the composition compared to the bactericidal titer in the human before administration of the dose, when measured under the same conditions in an hSBA.

[0198] In one embodiment, a "responder" refers to a human in whom a composition induces a bactericidal titer after administration of a dose, the bactericidal titer being at least one-fold greater than the bactericidal titer in the human before administration of the dose. In preferred embodiments, the responder achieves at least a four-fold or greater increase in hSBA titer compared to the bactericidal titer in the human before administration of the dose. Such a responder may be said to have a protective titer. In some embodiments, a protective titer is greater than 1:4.

[0199] In one embodiment, the hSBA titer is the reciprocal of the highest dilution of a serum sample that produces a measurable effect. For example, in one embodiment, the hSBA titer is the reciprocal of the highest 2-fold dilution of test serum that results in at least a 50% reduction in MnB bacteria (50% bacterial survival) compared to the T30 CFU value (i.e., the number of bacteria surviving after incubation in an assay well containing all assay components except test serum; 100% bacterial survival).

[0200] In one embodiment, the composition induces a bactericidal titer in a human after receiving the first dose that is at least two-fold higher than the bactericidal titer in the human before receiving the first dose, as measured under identical conditions in an hSBA (e.g., higher than the bactericidal titer in the human in the absence of the first dose). In one embodiment, the composition induces a bactericidal titer in a human that is at least four-fold higher than the bactericidal titer in the human before receiving the first dose, as measured under identical conditions in a human serum bactericidal assay (hSBA) utilizing human complement. In one embodiment, the composition induces a bactericidal titer in a human that is at least 8-fold higher than the bactericidal titer in the human before receiving the first dose, when measured under identical conditions in a human serum bactericidal assay (hSBA) utilizing human complement.

[0201] In a preferred embodiment, the human serum complement is derived from a human having low intrinsic bactericidal activity for a given hSBA test strain. Low intrinsic bactericidal activity refers, for example, to a bactericidal titer that is at least less than a 1:4 dilution for a given hSBA test strain. In one embodiment, the human complement is derived from a human having an hSBA titer that is at least less than a 1:4 dilution, such as 1:2, for a given hSBA test strain, and the human has not been administered a composition.

[0202] A human may exhibit an hSBA titer of less than 1:4 prior to administration of a composition, such as a bivalent rLP2086 composition, or a human may exhibit an hSBA titer of 1:4 or greater prior to administration of the composition. Thus, in preferred embodiments and examples, administration of at least one dose of a composition to a human results in an hSBA titer that is at least four-fold higher than the titer in the human prior to administration. In some embodiments, administration of at least one dose of a composition to a human results in an hSBA titer of at least 1:4, such as an hSBA titer of 1:8 or greater, an hSBA titer of 1:16 or greater, and an hSBA titer of 1:32 or greater. Each example described herein includes an assessment of the proportion of human subjects with hSBA titers of 1:8 or greater and / or 1:16 or greater, and the bivalent rLP2086 composition was administered to the human. In some embodiments, a four-fold increase in titer in a human after administration of the composition compared to before administration indicates that protection is associated with the composition. In some embodiments, such a favorable assessment of hSBA titer greater than 1:4 indicates that protection, ie, the induction of a bactericidal immune response in humans, is associated with the composition.

[0203] In one embodiment, the human has an hSBA titer at or above the lower limit of quantitation (LLOQ) for the hSBA after administering a first dose of the composition. In another embodiment, the human has an hSBA titer at or above the LLOQ for the hSBA after administering a second dose of the composition. In another embodiment, the human has an hSBA titer at or above the LLOQ for the hSBA after administering a third dose of the composition. Methods and Administration In one aspect, the disclosure relates to a method of inducing an immune response against meningococcus in a human, hi another aspect, the disclosure relates to a method of vaccinating a human.

[0204] In some aspects, the method comprises administering a composition to a human, wherein the composition induces an immune response against each of meningococcal serotypes A, C, W-135, and Y capsular polysaccharides, wherein the immune response comprises a titer of serum bactericidal antibodies that is higher than that induced by a licensed vaccine against meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharides. In some aspects, the licensed vaccine against meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharides is MENVEO.

[0205] In some aspects, the method includes administering a composition to a human, wherein the composition induces an immune response against meningococcal serogroup B, wherein the immune response comprises a titer of serum bactericidal antibodies that is higher than the titer of serum bactericidal antibodies induced by a licensed vaccine against meningococcal serogroup B. In some aspects, the licensed vaccine against meningococcal serogroup B is TRUMENBA.

[0206] In some aspects, the method comprises administering a composition to a human, wherein the composition induces an immune response against each of meningococcal serotypes A, C, W-135, and Y capsular polysaccharides and meningococcal serotype B, wherein the immune response comprises titers of serum bactericidal antibodies against each of meningococcal serotypes A, C, W-135, and Y capsular polysaccharides that are higher than the titers of serum bactericidal antibodies induced by licensed vaccines against meningococcal serotypes A, C, W-135, and Y capsular polysaccharides. and the immune response comprises a titer of serum bactericidal antibodies against meningococcal serogroup B that is higher than the titer of serum bactericidal antibodies induced by a licensed vaccine against meningococcal serogroup B. In some embodiments, the licensed vaccine against meningococcal serogroups A, C, W-135, and Y meningococcal capsular polysaccharide is MENVEO. In some embodiments, the licensed vaccine against meningococcal serogroup B is TRUMENBA.

[0207] The present disclosure relates to methods of eliciting an immune response in humans of any age. In some embodiments, the human is at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks old. For example, in a preferred embodiment, the human is at least 6 weeks old. As known in the art, meningococcal A, C, W-135, and Y conjugate vaccines, such as NIMENRIX®, are suitable for infants as early as 6 weeks old and can be administered to any human 6 weeks or older. In some embodiments, the human is at least 6, 7, 8, 9, 10, 11, or 12 months old. For example, in a preferred embodiment, the human is at least 12 months old. In one embodiment, the human is 12 to 18 months old. In another aspect, the disclosure relates to a method of inducing an immune response in a patient at least 18 months of age. In one aspect, the human is 18 to 24 months of age. In yet another aspect, the disclosure relates to a method of inducing an immune response in a patient at least 24 months of age. In one aspect, the human is 24 months to 10 years of age. In another aspect, the disclosure relates to a method of inducing an immune response in a patient 10 years of age or older.

[0208] In some embodiments, the human is between 10 and 25 years old. In some embodiments, the human is between 10 and 26 years old. In some embodiments, the human is between 12 months and under 18 months old. In some embodiments, the human is between 18 months and under 24 months old. In some embodiments, the human is between 18 months and under 24 months old. In some embodiments, the human is between 24 months and under 10 years old.

[0209] In some embodiments, the human is at least 16 years of age. In such embodiments, the method comprises administering one dose to the human, preferably at most one dose to a human who is at least 16 years of age. In some embodiments, the human is at most 17 years of age.

[0210] In some embodiments, the human is 10-12 years of age. In such embodiments, the method comprises administering at least one dose to the human. In a preferred embodiment, a second dose is administered to the human about 6 months after the first dose. In a preferred embodiment, the method comprises administering a first and second dose of the composition to a human aged 10-12 years, and administering a third dose of the composition to the human at least 4 years after the first dose.

[0211] In some embodiments, the method includes administering at least two doses to a human. In preferred embodiments, the two doses are at least about six months apart. In preferred embodiments, the method includes administering a first and second dose of the composition to a human between the ages of 10 and 12 years, and administering a third dose of the composition to the human at least four years after the first dose.

[0212] In some embodiments, the method includes administering a first dose of the composition to a human at about age 11, and administering at least two doses of the composition to the human at least four years after the first dose. In some embodiments, the method includes administering a second and subsequent dose of the composition about five years after the first dose.

[0213] In some embodiments, the human is seronegative for meningococcal serogroup A. In some embodiments, the human is seronegative for meningococcal serogroup C. In some embodiments, the human is seronegative for meningococcal serogroup B. In some embodiments, the human is seronegative to meningococcal serogroup W. In some embodiments, the human is seronegative to meningococcal serogroup Y. In some embodiments, the human is seronegative to meningococcal serogroups A, C, W-135, and Y capsular polysaccharides.

[0214] In some embodiments, the human is seropositive for meningococcal serotype A. In some embodiments, the human is seropositive for meningococcal serotype C. In some embodiments, the human is seropositive for meningococcal serotype W. In some embodiments, the human is seropositive for meningococcal serotype Y. In some embodiments, the human is seropositive for meningococcal serotypes A, C, W-135, and Y capsular polysaccharides.

[0215] In one embodiment, the method comprises administering at least one dose of the composition described above to a human. In a preferred embodiment, the method comprises administering at most one dose of the composition described above to a human. In another embodiment, the method comprises administering at least a first dose and a second dose of the composition described above to a human.

[0216] In one embodiment, the second dose is administered at least 20, 30, 50, 60, 100, 120, 160, 170, or 180 days after the first dose, and up to 250, 210, 200, or 190 days after the first dose. Any minimum value can be combined with any maximum value described herein to define a range.

[0217] In another embodiment, the second dose is administered about 30 days after the first dose. In another embodiment, the second dose is administered about 60 days after the first dose, such as, for example, in a 0.2 month immunization schedule. In another embodiment, the second dose is administered about 180 days after the first dose, such as, for example, in a 0.6 month immunization schedule. In yet another embodiment, the second dose is administered about 120 days after the first dose, such as, for example, in a 2.6 month immunization schedule.

[0218] In one embodiment, the method comprises administering two doses of the composition to a human, and at most two doses. In one embodiment, the two doses are administered within about six months of the first dose. In one embodiment, the method does not comprise further administration of a booster to the human. As used herein, "booster" refers to an additional administration of the composition to a human. Administering at most two doses of the composition to a human can be advantageous. Such advantages include, for example, facilitating compliance of the human with the complete administration schedule and facilitating cost-effectiveness of the schedule.

[0219] In one embodiment, the first and second doses are administered to a human over a period of about 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days, and up to 400, 390, 380, 370, 365, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, or 200 days after the first dose. Any minimum value may be combined with any maximum value described herein to define a range. Preferably, the first and second doses are administered at least four weeks apart, e.g., eight weeks or more apart, two months or more apart, three months or more apart, six months or more apart, etc.

[0220] In one embodiment, the first and second doses are administered to a human over a period of about 30 days. In another embodiment, the first and second doses are administered to a human over a period of about 60 days. In another embodiment, the first and second doses are administered to a human over a period of about 180 days.

[0221] Advantageously, the first dose can be administered substantially simultaneously with another vaccine (e.g., during the same medical consultation or visit to a health care professional, or within 24 hours of the first dose of meningococcal vaccine), for example, hepatitis B virus vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine (either cellular or preferably acellular), Haemophilus influenzae type b vaccine, pneumococcal vaccine, and / or polio vaccine (preferably inactivated poliovirus vaccine). Each of these optionally co-administered vaccines can be a monovalent vaccine or can be part of a combination vaccine (e.g., as part of a DTP vaccine).

[0222] Advantageously, the second dose may be administered substantially simultaneously with another vaccine (e.g., during the same medical consultation or visit to a health care professional or within 24 hours of the second dose of meningococcal vaccine), such as hepatitis B virus vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine (either cellular or acellular), Haemophilus influenzae type b vaccine, pneumococcal vaccine, polio vaccine (preferably an inactivated poliovirus vaccine), influenza vaccine, chickenpox vaccine, measles vaccine, mumps vaccine, and / or rubella vaccine. Each of these optionally co-administered vaccines may be a monovalent vaccine or may be part of a combination vaccine (e.g., as part of an MMR vaccine).

[0223] Advantageously, the third dose may be administered substantially simultaneously with another vaccine (e.g., during the same medical consultation or visit to a health care professional or within 24 hours of the third dose of meningococcal vaccine), such as hepatitis B virus vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine (either cellular or acellular), Haemophilus influenzae type b vaccine, pneumococcal vaccine, polio vaccine (preferably an inactivated poliovirus vaccine), influenza vaccine, chickenpox vaccine, measles vaccine, mumps vaccine, and / or rubella vaccine. Each of these optionally co-administered vaccines may be a monovalent vaccine or may be part of a combination vaccine (e.g., as part of an MMR vaccine).

[0224] In one aspect, a three-dose schedule of the composition induces bactericidal titers against multiple strains expressing LP2086 (fHBP) heterologous to the first and / or second polypeptide in a higher percentage of humans than a two-dose schedule.

[0225] In one aspect, the method comprises administering three doses of the composition to the human. In another aspect, the method comprises administering up to three doses of the composition. In one aspect, the three doses are administered within a period of about six months or less after the first dose. In one aspect, the method comprises administering a booster dose to the human after the third dose. In another aspect, the method does not comprise administering a booster dose to the human after the third dose. In another aspect, the method does not further comprise administering a fourth or booster dose of the composition to the human. In a further aspect, up to three doses are administered to the human within a period of about six months or less.

[0226] In an exemplary embodiment, the second dose is administered about 30 days after the first dose and the third dose is administered about 150 days after the second dose, e.g., in a 0, 1, 6 month immunization schedule. In another exemplary embodiment, the second dose is administered about 60 days after the first dose and the third dose is administered about 120 days after the second dose, e.g., in a 0, 2, 6 month immunization schedule.

[0227] In one embodiment, a human is administered a first dose, a second dose, and a third dose over a period of about 150, 160, 170, or 180 days, and up to 240, 210, 200, or 190 days. Any minimum value may be combined with any maximum value described herein. The range can be defined by combining the above.Preferably, the first dose, the second dose and the third dose are administered to humans over a period of about 180 days or 6 months.For example, the second dose can be administered to humans about 60 days after the first dose, and the third dose can be administered to humans about 120 days after the second dose.Therefore, an exemplary administration schedule includes administering doses to humans at about 0, 2 and 6 months.

[0228] As noted above, multiple doses of the immunogenic composition may be administered to a human, and the number of days between each dose may vary. Advantages of this method include, for example, the flexibility it allows a human to comply with the administration schedule.

[0229] In one aspect, the method comprises administering to a human up to three doses of the same immunogenic composition. For example, in a preferred aspect, the method does not comprise administering to a human a first dose of a first composition, a second dose of a second composition, and a third dose of a third composition, where the first, second, and third compositions are not the same. In another aspect, the method comprises administering to a human up to four doses of the same immunogenic composition. [Example]

[0230] The following examples illustrate aspects of the present disclosure. Unless otherwise noted herein, the following examples refer to a MnB bivalent rLP2086 composition at a dose level of 120 μg of bivalent rLP2086, which comprises, per 0.5 mL dose, 60 μg of a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, 60 μg of a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, a molar ratio of polysorbate-80 to first polypeptide of 2.8, a molar ratio of polysorbate-80 to second polypeptide of 2.8, and 0.5 mg of Al. 3+ 10 mM histidine and 150 mM sodium chloride.

[0231] More specifically, the bivalent recombinant rLP2086 vaccine of this study at a dose level of 120 μg bivalent rLP2086 contains (a) 60 μg of a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, (b) 60 μg of a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, (c) 18 μg of polysorbate-80, (d) 250 μg of aluminum, (e) 780 μg of histidine, and (f) 4380 μg of sodium chloride. Each dose is 0.5 mL.

[0232] Unless otherwise noted herein, the following examples refer to the MenACWY-TT composition, which is a preferred exemplary embodiment of a tetravalent meningococcal polysaccharide conjugate composition comprising meningococcal capsular polysaccharides A, C, W-135, and Y, each coupled to tetanus toxoid as a carrier protein. The meningococcal serogroup A and C polysaccharides are conjugated with an adipic acid dihydrazide (AH) spacer and indirectly conjugated to tetanus toxoid, while the W-135 and Y polysaccharides are directly conjugated to tetanus toxoid. The composition does not contain any preservatives or adjuvants.

[0233] More specifically, the lyophilized MenACWY-TT composition described in the Examples below contains, per dose (0.5 mL), 5 micrograms of meningococcal serogroup A polysaccharide conjugated to a tetanus toxoid carrier protein, 5 micrograms of meningococcal serogroup C polysaccharide conjugated to a tetanus toxoid carrier protein, 5 micrograms of meningococcal serogroup W-135 polysaccharide conjugated to a tetanus toxoid carrier protein, 5 micrograms of meningococcal serogroup Y polysaccharide conjugated to a tetanus toxoid carrier protein, 28 mg of sucrose, and 97 μg of trometamol. [Example]

[0234] MenABCWY composition The final MenABCWY composition is prepared by reconstituting a vial of lyophilized MenACWY-TT drug product (described in Example 2 below) with 0.67 mL of MnB bivalent rLP2086 drug product (described in Example 3 below) to derive a 0.5 mL dose of MenABCWY vaccine for intramuscular injection. All components used in the preparation of the MenABCWY vaccine and their functions are provided in Table 1 below.

[0235] [Table 1]

[0236] MenABCWY is a combination product containing a single dose of lyophilized MenACWY-TT in a 2 mL Type 1 glass vial and an ungraded 1 mL Type I glass standard pre-filled syringe (PFS) containing MnB bivalent rLP2086 suspension for reconstitution, along with a 13 mm vial adapter.

[0237] Lyophilized MenACWY-TT drug product lyophilized vial adapter supplied The PFS of MnB bivalent rLP2086 is reconstituted by securing it to a vial and attaching it to a vial adapter. This process is intended to facilitate aseptic transfer of the MnB suspension from the PFS into the vial, followed by transfer of the reconstituted vaccine from the vial into the PFS for administration. After reconstitution, the contents of the vial are drawn into the same syringe through the vial adapter. The vial adapter is removed from the syringe, and a needle is secured to the PFS for intramuscular (IM) injection. To ensure that the target dose volume (0.5 mL) of the final vaccine (MenABCWY) can be delivered, the PFS of MnB bivalent rLP2086 is designed to have a higher fill volume than TRUMENBA (approximately 0.67 mL versus the current 0.57 mL).

[0238] The final vaccine composition contains rLP2086 subfamily A and B proteins formulated at 120 μg / mL / subfamily in 10 mM histidine, 1.2 mM Tris buffer, 160.5-161.1 mM sodium chloride, 0.5 mg / mL aluminum as aluminum phosphate (AlPO), 1.35 mg / mL polysorbate 80, and 56 mg / mL sucrose, and purified capsular polysaccharides of meningococcal serotypes A, C, W, and Y conjugated to tetanus toxoid at ratios of about 1:3, about 1:3, about 1:1.5, and about 1:1.3, respectively, at a concentration of 10 μg / mL / type. [Example]

[0239] MnB bivalent rLP2086 drug product description and composition The MnB bivalent rLP2086 drug product is a sterile liquid formulation consisting of rLP2086 subfamily A and B proteins formulated at 120 μg / ml / subfamily in 10 mM histidine buffer, 150 mM sodium chloride (NaCl), pH 6.0, with 0.5 mg / mL aluminum as aluminum phosphate (AlPO4). Polysorbate 80 (PS-80) is added to the drug substance to achieve the target PS-80-to-protein molar ratio. Therefore, PS-80 is not added during drug product formulation; it is present in the final drug product at the same ratio. The drug product is filled into 1 mL syringes. A single dose of the vaccine is 0.5 mL and preservative-free.

[0240] [Table 2]

[0241] Effect of polysorbate 80 concentration Polysorbate 80 (PS-80) is a nonionic surfactant used to stabilize and solubilize MnB rLP2086 subfamily A and B proteins in formulations by preventing aggregation and adsorption, which can be caused by temperature, filters, tubing, container / closure contact, and process mixing. Stability studies using an in vitro monoclonal antibody-based potency assay demonstrated that subfamily B proteins are unstable at higher PS-80 to MnB rLP2086 protein molar ratios in the final formulation. Experiments using various PS-80 to protein molar ratios demonstrated that the optimal PS-80 to MnB rLP2086 protein molar ratio for retaining potency is approximately 1.4-4.2. [Example]

[0242] Description and composition of MenACWY-TT composition The MenACWY-TT drug product consists of purified polysaccharides of meningococcal serotypes A, C, W, and Y conjugated to tetanus toxoid (TT) at ratios of about 3, about 3, about 1.5, and about 1.3 to polysaccharide, respectively.

[0243] MenACWY-TT drug product is presented as a lyophilized powder supplied in 3 mL glass vials with bromobutyl rubber closures and aluminum flip-off caps suitable for lyophilization. All components used in the manufacture of MenACWY-TT drug product and their functions are provided in Table 3.

[0244] [Table 3] [Example]

[0245] Preparation of MenABCWY Composition The final MenABCWY composition is prepared in the clinic by reconstituting a lyophilized MenACWY-TT drug product vial with 0.67 mL of MnB bivalent rLP2086. The resulting MenABCWY composition (vaccine liquid drug product) is prepared for intramuscular injection in 10 mM histidine and 1.6 mM Tris buffer containing 160.5-161.1 mM sodium chloride, 0.5 mg / mL aluminum phosphate (AlPO4), and 0.5 mg / mL aluminum phosphate (AlPO4). The formulation contains rLP2086 subfamily A and B proteins at 120 mcg / mL / subfamily and purified meningococcal serotypes A, C, W, and Y polysaccharides at a concentration of 10 mcg / mL / type conjugated to tetanus toxoid in ratios of about 3, about 3, about 1.5, and about 3, respectively, in a 100% ethanol solution containing 0.035 mg / mL polysorbate 80, and 56 mg / mL sucrose, pH 6.05.

[0246] The MenABCWY vaccine is prepared by mixing two drug products, MenACWY-TT and MnB bivalent rLP2086. Buffer components and excipients are selected based on the individual development of each component and shown to provide the necessary stability profile for long-term shelf life.

[0247] A dosage validation study was conducted to demonstrate that the MenACWY-TT drug product and the MnB bivalent rLP2086 drug product were compatible when mixed together for administration of the MenABCWY vaccine, that all drug products and dosing solutions were compatible with the dosing components, and that the dosing solutions were stable in the dosing components for a period of time sufficient to perform dose preparation and administration procedures. The stability of the MenABCWY vaccine prepared by reconstitution of the MenACWY-TT drug product with 0.67 mL of the MnB bivalent rLP2086 drug product over the hold period at room temperature and in light was confirmed in the reconstituted vial and in the dosing syringe.

[0248] Samples representing the MenABCWY vaccine dosing solution were tested using stability-indicating methods, including RP-HPLC for antigen binding and purity, bioplex activity assay, ELISA, and ICP-MS using pre-determined acceptance criteria. The results of this study demonstrate acceptable stability of the MenABCWY vaccine for 24 hours at room temperature and in light. [Example]

[0249] Evaluation of the MenABCWY vaccine A study was conducted to evaluate whether acceptable physical compatibility and short-term stability existed when a lyophilized MenACWY-TT composition was reconstituted with a MnB bivalent rLP2086 composition. The lyophilized MenACWY-TT and liquid MnB bivalent rLP2086 compositions were combined and stored for up to 24 hours in an uncontrolled room temperature environment to mimic real-world conditions. It was demonstrated that the lyophilized MenACWY-TT composition could be reconstituted with the liquid MnB bivalent rLP2086 composition with gentle manual mixing, and the combined pH and osmolality were within the typical ranges for injectable formulations. All key attributes of the conjugate and protein were similar to those of the control for up to 24 hours in an uncontrolled room temperature environment.

[0250] Physical compatibility was assessed by evaluating the combined drug product pH, appearance, ease of resolubilization, and osmolality. Antigen stability was assessed by assessing the concentration, purity, and in vitro relative antigenicity (IVRA) of rLP2086 subfamily A and subfamily B proteins, and the concentrations of conjugated meningococcal A, C, Y, and W-135 polysaccharides by ELISA.

[0251] Examples 5 through 15 demonstrate that the combination of a lyophilized MenACWY-TT composition and a liquid MnB bivalent rLP2086 composition, i.e., a MenABCWY composition, was found to be compatible and stable at room temperature for at least 24 hours. ELISA for determining the concentration of Mening A, C, Y, and W-135 polysaccharides in MenABCWY compositions - Development of ELISA for Mening A, C, Y, and W-135 and screening of pAbs for detection Six antibodies were selected for screening for use in ELISA assays. Four groups of 10 rabbits each were immunized with either Men A, C, Y, or W-135 polysaccharide TT conjugates, followed by exsanguination of the rabbits after antibody development. Each rabbit was immunized with the carrier protein CRM for binding and specificity. 197Rabbit sera were individually screened using Men A, C, Y, or W-135 polysaccharide conjugated with the CRM conjugate. Rabbit sera were screened for a positive binding signal, equivalent to an absorbance reading greater than three times background absorbance. Additionally, rabbit sera were screened for low nonspecific binding, defined as any absorbance reading of a serum combination without antigen, secondary antibody, or detection antibody greater than the blank absorbance reading, and low cross-reactivity, defined as any absorbance reading of a nonhomologous serotype greater than background absorbance. Rabbits that met the screening criteria were pooled. A calibration curve range was established using the CRM conjugate and confirmed with reconstituted lyophilized MenACWY-TT composition. A calibration curve range was established using the CRM conjugate and confirmed with reconstituted lyophilized MenACWY-TT composition.

[0252] The feasibility of quantifying A, C, Y, and W conjugates in a combined drug product (MenABCWY composition) was established. It was determined that the MnB bivalent rLP2086 composition alone was not detected by the assay. Furthermore, complete recovery of the conjugates was obtained when aluminum phosphate in the MenABCWY composition sample was solubilized. Therefore, it was determined that the MnB bivalent rLP2086 composition does not interfere with the quantification of MenABCWY-TT conjugates by ELISA. [Example]

[0253] Evaluating the suitability of the method to evaluate the MnB bivalent rLP2086 composition in the presence of the MenACWY-TT composition IEX-HPLC was evaluated for its suitability for determining the intensity of MnB bivalent rLP2086 subfamily A and B proteins in the presence of MenACWY-TT. Total and bound protein results for MnB bivalent rLP2086 in the presence and absence of MenACWY-TT were evaluated.

[0254] [Table 4] [Example]

[0255] Evaluation of the purity and peak ratio of MnB divalent rLP2086 composition in the presence of MenACWY-TT composition RP-HPLC was evaluated for its suitability for determining the purity of the MnB bivalent rLP2086 composition in the presence of MenACWY-TT composition. The purity results of the MnB bivalent rLP2086 composition in the presence and absence of MenACWY-TT composition were compared. The overlaid chromatograms are shown in Figure 2 of U.S. Patent No. 10,183,070. An example of impurity peak integration is shown as an inset in Figure 2 of U.S. Patent No. 10,183,070. The evaluation results indicate that the presence of MenACWY-TT composition does not interfere with the evaluation of the purity of the MnB bivalent rLP2086 composition using the RP-HPLC method. [Example]

[0256] IVRA evaluation of MnB bivalent rLP2086 composition in the presence of MenACWY-TT composition The IVRA method was evaluated for its suitability to determine the in vitro relative antigenicity of the MnB bivalent rLP2086 composition subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) proteins in the presence of the MenACWY-TT composition.

[0257] We compared the results of IVRA of MnB bivalent rLP2086 subfamily A and subfamily B proteins in the presence and absence of MenACWY-TT composition. Feasibility assessment results indicate that within assay variability, results are comparable and that the presence of MenACWY-TT composition does not interfere with the determination of in vitro relative antigenicity. [Example]

[0258] Reconstitution of a vial of MenACWY-TT composition with the MnB bivalent rLP2086 composition Tests for the MenACWY-TT and MnB bivalent rLP2086 drug products were performed using vials of the MenACWY-TT composition reconstituted with the MnB bivalent rLP2086 drug product. Vials of the MenACWY-TT composition reconstituted with either saline or the MenACWY-TT composition were used as controls, with matrix placebo depending on the method.

[0259] [Table 5]

[0260] Determining the reconstitution volume of saline for MenACWY-TT compositions The NIMENRIX® commercial product package contains both a vial containing the lyophilized MenACWY-TT composition and a syringe containing 0.9% saline for use in reconstitution. To replicate the final NIMENRIX® concentration in the commercial vaccine upon reconstitution with the MnB bivalent rLP2086 composition, it was necessary to determine the amount of saline dispensed from the commercial product using the syringe. This same volume of MnB bivalent rLP2086 composition was then used for all reconstitution studies. Reconstitution of a vial of MenACWY-TT composition with the MnB bivalent rLP2086 composition The MnB bivalent rLP2086 composition was pooled in a 10 mL glass vial. Approximately 800 μL of the solution was drawn into a 1 mL syringe. The adjusted contents of the syringe were injected into the vial containing the MenACWY-TT composition. The vial was swirled to dissolve the contents.

[0261] The pH and appearance of duplicate samples of the MenACWY compositions were determined. Osmolality was measured in triplicate for MenACWY-TT compositions reconstituted with saline and for MenACWY-TT compositions reconstituted with the MnB bivalent rLP2086 composition. [Example]

[0262] SEC-MALLS to evaluate the stability of Mening A, C, Y, and W-135 polysaccharides in DP matrices. Meningococcal A, C, Y, and W-135 polysaccharides were used as a surrogate to assess whether any instability of the conjugated meningococcal A, C, Y, and W-135 polysaccharides in the combined drug product (MenABCWY composition) could be expected. Processing of Mening A, C, Y, and W-135 polysaccharides Preparation of Reagent ("Complete MenABCWY Composition Buffer Matrix") 2.24 g of sucrose and 7.8 mg of Tris(tromethamine) were added to 20 ml of 2x MnB bivalent rLP2086 composition buffer matrix along with MnB rLP2086 protein (20 mM histidine, pH 6.0, 300 mM NaCl, 0.07 mg / ml PS80, 1 mg / ml (8 mM) AlPO4, 240 μg / mL each of rLP2086 subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) proteins). Sample preparation Each Mening polysaccharide was diluted 1:1 with the complete MenABCWY composition buffer matrix and incubated for 0, 6, and 24 hours at 5°C, 25°C, and 37°C. After incubation, the sample suspension was centrifuged for 1 minute at 14,000 rpm. The supernatant was analyzed by SEC-MALLS. [Example]

[0263] Stability of MenABCWY Compositions - Evaluation of pH, Appearance, and Osmolality of Combined MnB Bivalent rLP2086 and MenACWY-TT Compositions The pH and appearance of the combined MnB bivalent rLP2086 and MenACWY-TT compositions, ie, the MenABCWY compositions, were evaluated immediately after reconstitution and again after 24 hours.

[0264] All results were as expected (Table 6).

[0265] [Table 6]

[0266] The mean osmolality of the MenACWY-TT composition reconstituted with the MnB divalent rLP2086 composition was within 3% of the mean osmolality of the MenACWY-TT composition reconstituted with saline.

[0267] [Table 7] [Example]

[0268] Concentrations of Mening A, C, Y, and W-135 polysaccharide conjugates in the combined drug product The concentrations of Mening A, C, Y, and W-135-TT conjugates in the MenABCWY composition were evaluated initially and again after 24 hours. was stable over the 24-hour period (Table 8).

[0269] [Table 8] [Example]

[0270] Assessment of the stability of MnB bivalent rLP2086 protein in MenABCWY compositions Concentration of total and bound rLP2086 subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) proteins in the combined drug product Samples of the MenABCWY composition were analyzed by IEX-HPLC to determine protein concentration. As shown in Table 9, the total protein, bound protein (with aluminum), and % bound for both the MnB bivalent rLP2086 subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) proteins (with aluminum) did not change within 24 hours, indicating that the rLP2086 subfamily A and subfamily B proteins were stable over the 24 hour period.

[0271] [Table 9] [Example]

[0272] Purity and peak ratio of rLP2086 protein in the combined MenABCWY composition A sample of the MenABCWY composition was analyzed by RP-HPLC to determine the purity and peak ratio of the rLP2086 protein. See Figure 3 of U.S. Patent No. 10,183,070. The peak at 11.9 minutes is excluded from the purity calculation. IVRA of rLP2086 subfamily A and subfamily B proteins in combined drug products The IVRA of samples of the MenABCWY composition was evaluated up to 24 hours after mixing. It was determined that the relative antigenicity of the rLP2086 subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) proteins in the MenABCWY composition was stable over the 24 hour period. [Example]

[0273] SEC-MALS analysis of Mening in the complete MenABCWY composition buffer matrix Stability of A, C, Y, and W-135 polysaccharides Stability of Mening A PS in the complete MenABCWY composition buffer matrix by SEC-MALLS after 6 and 24 hours of incubation at various temperatures Mening A, C, W, and Y polysaccharides were mixed with the complete MenABCWY composition buffer matrix and evaluated for stability by SEC-MALS after incubation at 5°C, 25°C, and 37°C for up to 24 hours. All four polysaccharides appear stable for up to 24 hours at 5°C and 25°C. Some degradation was observed for Mening A and Y at 37°C. The extent of degradation could not be determined for Mening Y polysaccharide due to the formation of high Mw aggregates under all but the initial conditions tested.

[0274] [Table 10] [Example]

[0275] Evaluation of meningococcal serogroup B immunogenicity of Mn pentavalent and Trumenba® vaccines in CBA / J mice The immune response to meningococcal serogroup B fHBP after vaccination with either the bivalent Mn B fHBP vaccine, Trumenba, or the bivalent Mn B fHBP vaccine formulated with the tetravalent ACWY polysaccharide conjugate vaccine (Mn pentavalent ABCWY) was evaluated in CBA / J mice. Groups of CBA / J mice were immunized with three different vaccines: pentavalent (ABCYW), Trumenba® (MnB), and Nimenrix® (ACYW) (Table 11).

[0276] [Table 11]

[0277] CBA / J mice (25 / group) were immunized subcutaneously in the scruff of the neck with two-fold dilutions of each vaccine dose level for each arm (Table 11). Mice were primed with vaccine at time 0 and boosted at week 2. Serum was collected at week 3 on PD2 for testing using two different human complement-based serum bactericidal assays (hSBA). One hSBA used an fHBP subfamily A-expressing strain (M98250771), and the other used an fHBP subfamily B-expressing strain (CDC1127).

[0278] The hSBA measures antibody-dependent, complement-mediated bactericidal activity against meningococcal serogroup B strains. Briefly, appropriate dilutions of test serum were mixed in a 96-well microtiter assay plate with a freshly prepared bacterial culture of meningococcal B strains (subfamily A or B) and human complement. The assay plate was placed on an orbital shaker and mixed for 30 minutes in a humidified incubator (37°C / 5% CO). An aliquot of the assay reaction from each well was then transferred to a 96-well filter plate for enumeration of surviving bacteria.

[0279] Response rates to vaccination were calculated as the percentage of mice in each dosing group (n=25) that responded to hSBA. 30 Mouse serum samples that kill 50% or more of the control meningococcal bacteria are considered responders. 30 Control wells contain bacteria and complement but no test serum and are counted at the end of the 30 minute assay incubation. Table 12 and Table 11 Table 13 shows the comparative results induced by either TRUMENBA® or Mn pentavalent for both subfamily A and subfamily B meningococcal serogroup B strains. A possible dose-dependent response rate is shown. As expected, NIMENRIX™ did not induce a functional immune response against the Mn B strain.

[0280] [Table 12]

[0281] [Table 13] [Example]

[0282] A study to describe the immunogenicity, safety, and tolerability of a bivalent rLP2086-containing pentavalent vaccine (MenABCWY) in healthy subjects aged 10 to 26 years (B1971057) B1971057 is a Phase 2 proof-of-concept (POC) study to evaluate the safety and immunogenicity of the pentavalent in healthy subjects aged 10 to under 26. The study began in April 2017, and approximately 530 subjects received the pentavalent.

[0283] Meningococcal vaccines are licensed by the human complement serum bactericidal assay (hSBA), an immunological surrogate that demonstrates the ability of immune serum to kill meningococcal strains representing the serotypes included in the vaccine. For MenACWY responses, one strain from each serotype was evaluated in the hSBA, and pentavalent responses were compared to the licensed ACWY vaccine, MENVEO. For MenB evaluation, four serotype B strains that were also used during the TRUMENBA licensing study were used. Tested.

[0284] Study data showed that the pentavalent was non-inferior to Menveo after one vaccination for ACWY assessment and non-inferior to Trumenba after two vaccinations for B assessment. Pentavalent vaccine in this study The investigational drugs for this study are bivalent rLP2086 (TRUMENBA), (pentavalent, described above in Examples 1 and 4), MenACWY-CRM (MENVEO), and placebo. research design B1971057 is a phase 3, randomized, actively controlled, observer-blinded, multicenter trial that randomly assigned approximately 1,590 subjects to receive either pentavalent plus placebo (saline) or Trumenba (Pfizer) plus Menveo (GSK). All subjects were naive to any meningococcal B vaccine prior to enrollment. Randomization was stratified by prior vaccination history, with approximately 50% ACWY-naive subjects and approximately 50% ACWY-experienced subjects (those who had received one previous dose of a vaccine containing one or more ACWY types ≥4 years prior to the date of randomization). This ACWY-experienced group was included because 86% of teenagers in the United States receive one dose of ACWY vaccine at approximately age 11 and should receive a booster dose at age 16. Randomization was also stratified by geographic region. Approximately 80% of subjects were from U.S. study sites, and approximately 20% were from Europe. Geographic stratification ensured adequate population representation. The study was conducted in two phases. Phase 1, now completed, included the vaccination phase of the primary series. The visit schedule for Phase 1 is noted below in Table 14. Phase 2 evaluated the durability of immunization and the booster dose administered approximately 4 years after completion of the pentavalent primary series.

[0285] [Table 14]

[0286] To assess immune responses, functional antibodies were analyzed in the hSBA using meningococcal serotypes A, B, C, W, and Y strains. The hSBA measures antibodies in human serum that result in complement-dependent killing of target meningococcal strains. To assess immune responses to Trumenba and the pentavalent B component, four primary MnB test strains, namely, PMB80 (A22), PMB2001 (A56), PMB2948 (B24), and PMB2707 (B44), were used in the hSBA to determine immunogenicity endpoints in this study. To assess immune responses to MENVEO and the pentavalent ACWY component, test strains specific to each ACWY serotype were identified and characterized in the hSBA prior to study initiation. The validated assay used to assess MenB was the same as that used to license TRUMENBA. the purpose This study was designed to describe the safety, tolerability, and immunogenicity of the pentavalent and immune responses against serogroups A, B, C, W, and Y following administration of the US-licensed meningococcal A, C, W-135, and Y conjugate vaccines, Trumenba and Menveo. Nimenrix was not used as an ACWY comparator because it is not licensed in the US.

[0287] The immunogenicity of the pentavalent MenACWY components was compared to the hSBA GMT observed for Trumenba + Menveo after a single dose of pentavalent hSB after a single dose. A Based on geometric mean titer (GMT).

[0288] We performed this evaluation on individuals who had not previously received a single dose of MenACWY vaccine (ACWY-naive individuals) and individuals who would receive their second booster dose of MenACWY (ACWY-experienced individuals). ACWY-naive individuals were required to meet the POC criteria for the pentavalent MenACWY component.

[0289] The immunogenicity criteria for the pentavalent MenB component were based on estimates of the achievement point of a 4-fold increase and composite hSBA response after two doses of the pentavalent administered on a 0, 6-month schedule that would predict meeting the phase 3 LCI criteria established for the 0, 6-month schedule.

[0290] Secondary endpoints of the study included standard non-inferiority assessment of the ratio of the two GMTs with a 2.0-fold margin and percent responders for the pentavalent B component. Analysis of percent responders was performed by defining responders as those achieving a 4-fold or greater rise in hSBA titer for the primary MenB strain and a combined response 1 month after vaccination, and then calculating the difference between pentavalent and TrumenB. A difference of no more than 10% was required to achieve success. Immune response to pentavalent and POC immunogenicity results Below, Study B1971057 shows that with regard to the bactericidal response to MenACWY between pentavalent and Menveo, similar hSBA GMTs were observed for the ACWY component after a single dose in subjects receiving pentavalent compared to Menveo. A non-inferiority (NI) margin of 1.5x GMT ratio was achieved in ACWY-naive subjects (Table 15), and an NI margin of 2.0x GMT ratio was achieved in ACWY-experienced subjects (Table 16).

[0291] [Table 15]

[0292] [Table 16]

[0293] Trumenba was approved based on the proportion of subjects achieving a 4-fold rise in antibody titer (and combined response) that met a pre-specified lower 95% confidence interval (LCI) threshold. When assessing 4-fold antibody responses, these may be influenced by background titers in the population, which can independently affect the proportion of subjects with a 4-fold response to the vaccine. Furthermore, the hSBA assay used is composed of biological components, which, although tightly controlled, may affect absolute response criteria between studies. Given that the LCI threshold for approval was calculated based on point estimates achieved using different complement sources in different populations, we also compared the pentavalent serotype B responses with those from the Trumenba + Menveo arm. The results are provided in Table 17.

[0294] [Table 17]

[0295] For three of the four serogroup B test strains, the fourfold increase in SBA and combined response significantly exceeded the prespecified point estimate. For one strain, PMB80 (A22), 75.8% of subjects achieved a fourfold increase in titer compared with a POC criterion of 78.1% (point estimate (PE) of 73.8 in the Trumenba group). However, as noted above, a 95% LCI criterion is more difficult to control in different populations over time. The phase 2 study used to generate the LCI for this POC study enrolled a European population, with a baseline hSBA rate of 22.1% for A22. The higher PE of the pentavalent compared with Trumenba suggested that there was no immune interference of the pentavalent MenB component. To confirm this point, secondary endpoint analyses demonstrated that Trumenba + Menveo was noninferior to the pentavalent in two noninferiority analyses. Indeed, they met the stringent 1.5 GMR non-inferiority margin (Table 18) and the percent responder analysis at the 5% margin (Table 19).

[0296] [Table 18]

[0297] [Table 19] [Example]

[0298] Potential public health impact of a pentavalent vaccine targeting meningococcal serogroups A, B, C, W, and Y in the United States Objective: To assess the potential for further reduction in IMD cases among the US population under various assumptions of vaccination schedules and compliance rates using a pentavalent vaccine.

[0299] Vaccination scenario Four primary vaccination scenarios were analyzed and compared with the estimated number of cases averted under current recommendations and compliance levels (Figure 1). 1. Replace MenACWY / MenB vaccine with pentavalent at age 16 years and retain MenACWY at age 11 years. 2. Replacement of MenACWY / MenB vaccine with pentavalent at ages 11 and 16 years R 3. Substitute pentavalent MenACWY / MenB at age 16 years and no MenACWY vaccination at age 11 years 4. Replace MenACWY at age 11 years with a two-dose pentavalent and MenACWY / MenB at age 16 years with a single-dose pentavalent. To estimate the impact of the various recommendations on the overall level of IMD reduction, vaccination rates using each schedule were varied. Estimates of disease reduction were based on estimates of immunization rates among adolescents in the United States published in 2018.

[0300] - 86.6% received at least one dose of MenACWY - 68.1% of all adolescents received at least one dose of HPV - 50.8% received ≥2 doses of MenACWY - 17.2% received ≥1 dose of MenB - We assumed that coverage of the second dose in a two-dose series at age 16 years was 50% of the first dose 3 and 70% at age 11 years. result At current coverage levels and a vaccination schedule requiring a total of four injections, the MenACWY and MenB vaccines are estimated to have averted 178 IMD cases over 10 years compared with a hypothetical no vaccination at all. Replacing MenACWY and / or MenB vaccination with the pentavalent would eliminate at least one injection. Assuming immunization rates at age 16 remain similar to current MenACWY vaccination rates, the pentavalent vaccine was estimated to have averted a similar or greater number of IMD cases (Figure 3).

[0301] The two-dose pentavalent at the age of 11 years and the one-dose pentavalent at the age of 16 years (scenarios 10–12) prevented the most cases (up to 282) compared with other vaccination schedules with comparable coverage rates.

[0302] The one-dose pentavalent at age 11 years and the two-dose pentavalent at age 16 years (scenarios 4–6) could prevent up to 251 cases. At age 16 years, coverage with the second dose pentavalent is slightly higher than the current MenB coverage rate, so a similar number of IMD cases could be prevented with one dose of MenACWY vaccine at age 11 years and two doses of pentavalent at age 16 years (scenario 3) or two doses of pentavalent at age 16 years (scenario 8, see Figure 1). conclusion The disease impact of a vaccination strategy is directly related to the level of coverage achieved.

[0303] Replacing one or more doses of MenACWY or MenB vaccine with a pentavalent can further reduce IMD caused by all five meningococcal serotypes. reducing the number of vaccine doses administered to adolescents; Potentially improving compliance with ACIP recommendations and reducing the costs of medical visits and public health responses to individual IMD cases.

[0304] Invasive meningococcal disease (IMD), caused by Neisseria meningitidis, is a rare, rapidly progressive, potentially fatal infection, with the highest incidence observed in infants and adolescents. Serogroups A, B, C, W, and Y account for 94% of disease worldwide. In the United States, most disease is caused by serogroups B, C, and Y. According to 2018 US surveillance data, the incidence of IMD was 0.10 cases per 100,000 population. Among US adolescents and young adults (62% of cases among those aged 16–23 years) and Serotype B also predominated across all age groups (36% of cases).

[0305] The US Advisory Committee on Immunization Practices (ACIP) currently recommends two meningococcal vaccines to help protect healthy adolescents against IMD. The quadrivalent meningococcal serogroups A, C, W, and Y (MenACWY) vaccine is routinely recommended as a primary dose at age 11–12 years and a booster dose at age 16 years. Based on shared clinical decision-making, meningococcal serogroup B (MenB) vaccination is recommended for adolescents and young adults aged 16–23 years (preferably 16–18 years). In 2018, estimated MenACWY vaccination coverage among adolescents aged 13–17 years was 86.6% with one or more doses and 50.8% with two or more doses. In contrast, only 17.2% of 17-year-olds received one or more doses of MenB vaccine, and fewer than 50% of these individuals completed a multi-dose vaccination series. These data suggest that many adolescents in the United States are not fully protected against meningococcal disease.

[0306] Instead of two separate MenB and MenACWY vaccines with different vaccination schedules, a single vaccine (i.e., the MenABCWY pentavalent vaccine) that can help protect against meningococcal disease caused by all five serotypes could simplify immunization, reduce the number of injections required, and potentially improve vaccination coverage. We developed a model to assess the public health impact of different types of meningococcal immunization programs using the pentavalent MenABCWY vaccine. 2.0 Method 2.1 Model Description A population-based dynamic model was developed to estimate the expected number of IMD cases averted in the United States over a 10-year period. The model structure is similar to that previously described in detail elsewhere. The population was stratified into 101 single-year age bands, and individuals in each age band transitioned to the next age band the following year. Meningococcal carriage is the primary cause of infectious disease transmission and was the primary consideration in the model calculations. Meningococcal carriage and transmission were modeled by stratifying the population into 10 mutually exclusive age bands (0–5 months, 6–12 months, 1 year, 2–4 years, 5–9 years, 10–14 years, 15–19 years, 20–24 years, 25–59 years, and ≥60 years). Each age group was characterized by the proportion of individuals who were carriers of meningococcal serogroups A, B, C, W, and Y and had an age-specific probability of developing IMD and transmitting the bacteria within that age group or to other age groups. For each year, the proportion of meningococcal carriers within each of the 10 age groups was calculated based on: (1) the previous year's carriage prevalence, (2) bacterial transmission and mixing patterns within and between age groups, (3) the number of individuals vaccinated (vaccination coverage), and (4) vaccine effectiveness against carriage acquisition. During each year of the 10-year time horizon in the model, the proportion of individuals in the target age group receiving MenACWY, MenB, and / or MenABCWY vaccine under four different schedule scenarios was estimated. For all vaccination scenarios, the model estimated whether individuals who developed IMD recovered, with or without complications, or died. 2.2 Model Inputs For each serotype, mean age-group-based IMD incidence rates were derived from the Centers for Disease Control and Prevention (CDC) Enhanced Meningococcal Disease Surveillance Reports from 2015 to 2017. The MenABCWY vaccine was hypothesized to provide direct protection against serotypes A, B, C, W, and Y.

[0307] Serogroup B in adolescents receiving one or two doses of MenABCWY vaccine Vaccine efficacy assumptions against serogroup B were based on published clinical studies of the MenB-FHbp vaccine (Trumenba®, bivalent rLP2086, Pfizer Inc, Philadelphia, PA). In this study, the percentage of subjects with serum bactericidal activity titers ≥ 1:8 in a human complement-based assay (hSBA) 1 month after vaccination (the standard correlation for protection is ≥ 1:4) ranged from 23.8% to 67.6% and 69.1% to 100% after one or two doses of MenB-FHbp, respectively. Based on these data, estimates of 30% and 85% vaccine efficacy against serogroup B were assumed for adolescents receiving one or two doses of MenABCWY vaccine, respectively.

[0308] The vaccine efficacy assumption against serotypes A, C, W, and Y was based on a review of published immunogenicity and efficacy data from clinical studies of the MenACWY-TT vaccine (Nimenrix®, Pfizer Ltd, Sandwich, UK), in which the percentage of subjects with serum bactericidal activity titers ≥ 1:8 in rabbit complement-based assays (rSBA) or hSBA 1 month after vaccination ranged from 81.9% to 97.4% after a single dose of MenACWY-TT. Based on these data, an estimate of 95% vaccine efficacy against serotypes A, C, W, and Y was assumed.

[0309] Indirect protection in unvaccinated individuals due to reduced carriage prevalence and transmission was assumed to be 0% for serotype B and 36.2% for serotypes A, C, W, and Y, both derived from published literature for MenB and MenACWY vaccines.

[0310] The 5-year protection period and fixed 10% annual attenuation rate for the MenABCWY vaccine against serogroup B shown were assumed based on (1) a previously published health economic model and (2) consideration of clinical data from a phase 3 extension study in adolescents that evaluated the durability of the immune response elicited by the MenB-FHbp vaccine. Results from the clinical study showed that response rates peaked after primary vaccination, declined over the following 12 months, and then remained stable above baseline through 48 months. At 48 months after primary vaccination, 18.0% to 61.3% of subjects had hSBA titers at or above the lower limit of quantitation (i.e., 1:16 or 1:8, depending on the strain) across the four diverse serogroup B test strains used to assess breadth of protection.

[0311] For the evaluation of MenACWY vaccine protection against serotypes A, C, W, and Y (Table 2), a 5-year duration of direct protection and a 10% annual decline rate were conservative assumptions based on clinical data from a study evaluating the durability of immune responses induced by MenACWY-TT over 10 years after primary vaccination in adolescents and adults aged 11 to 55 years. At 10 years, 70.2%–90.7% of vaccinated subjects had rSBA titers ≥ 1:8 across serotypes A, C, W, and Y, compared with 99.7%–100% 1 month after vaccination. Currently, data are not available regarding the duration and decline rate of indirect protection for the licensed MenACWY vaccine. Therefore, in this model, we assumed that the decline rate of MenACWY was equal to that of MenABCWY. 2.3 Vaccination scenarios and sensitivity analyses Vaccination scenarios were constructed based on the existing adolescent meningococcal vaccination platform in the United States (i.e., ages 11 and 16 years). Four primary vaccination schedules were examined and compared with the current schedule: (1) one dose of MenACWY vaccine at age 11 years and two doses of MenABCWY vaccine at age 16 years, (2) one dose of MenABCWY vaccine at age 11 years and two doses of MenABCWY vaccine at age 16 years, (3) two doses of MenABCWY vaccine only at age 16 years, and (4) two doses of MenABCWY vaccine at age 11 years and one dose at age 16 years.

[0312] Vaccination coverage assumptions for each primary schedule were obtained from observed adolescent vaccination rates, age at vaccination, and number of doses required, as reported in the National Immunization Survey-Teen (NIS-Teen) 2018. Consistent with trends observed in the NIS-Teen survey, vaccination coverage among adolescents aged 11 years was assumed to be higher than at age 16 years, and compliance with the two-dose series (i.e., completion of the recommended dosing series) at age 11 years was assumed to be higher than compliance with the two-dose series at age 16 years.

[0313] For the base case analysis, first-dose vaccination coverage at age 11 years was assumed to be the same as the overall MenACWY primary dose coverage reported in 2018 (86.6%), and first-dose vaccination coverage at age 16 years was assumed to be the same as the MenACWY booster dose coverage (50.8%). Based on these data, it was assumed that 80% of adolescents at age 11 years who received one dose of MenABCWY would complete the two-dose series, while compliance with the two-dose series at age 16 years was assumed to be 50% based on available information on completion of the MenB vaccine series. Sensitivity analyses were conducted for each of the four meningococcal vaccine schedules using the adolescent vaccination coverage levels at age 16 years reported in 2018. The highest coverage assumed was the same as that for one or more doses of human papillomavirus (HPV) vaccine at the age of 11–12 years (68.1%), and the lowest coverage assumed was the same as that for one or more doses of MenB vaccine at the age of 16 years (17.2%). After considering the primary dosing schedule and vaccination coverage estimates, a total of 13 different scenarios were evaluated. 3.0 Results Based on the current vaccination schedule and reported vaccination rates in 2018 (MenACWY, 86.6% and 50.8% at age 11 and age 16 years, respectively; MenB, 17.2% at age 16 years), vaccination with two doses each of MenACWY and MenB vaccines, for a total of four injections at ages 11–16 years, could potentially avert 165 cases of IMD over the next 10 years compared with no meningococcal vaccination (the current scenario). Under this scenario, 19 serogroup B cases (11.5% of all preventable IMD cases) are estimated to be prevented over the next 10 years. Replacing either MenACWY and / or MenB vaccines with pentavalent MenABCWY vaccine would eliminate one or two injections, depending on the vaccination schedule, potentially averting a greater number of IMD cases (scenarios 1, 2, 4, 5, 7, 8, 10, and 11). This is assuming that MenABCWY vaccination rates at age 16 remain similar to MenACWY vaccination rates in 2018 (50.8%, scenarios 1, 4, 7, and 10) or possibly increase to the slightly higher HPV vaccination rates observed at ages 11–12 years (68.1%, scenarios 2, 5, 8, and 11).

[0314] However, assuming MenABCWY vaccination coverage at age 16 years is the same as the current two-dose MenB vaccination schedule at age 16 years (17.2%), a MenABCWY regimen of one dose at age 11 years and two doses at age 16 years (i.e., similar to the current schedule) would prevent fewer IMD cases (n = 137) compared to the current vaccination schedule (Figure 2, Scenario 6). These results are primarily driven by the assumption of lower MenABCWY vaccination coverage than currently reported at age 16 years, resulting in a lower estimated number of averted cases of serogroups A, C, W, and Y compared to the current schedule (n = 89, 65.0%). 3.1 Baseline case vaccination coverage assumptions In all base-case vaccination scenarios (scenarios 1, 4, 7, and 10), assuming MenABCWY vaccination rates equal the current coverage of MenACWY vaccine at age 11 years (86.6%) or age 16 years (50.8%), replacing either MenACWY and / or MenB vaccine with pentavalent MenABCWY vaccine would avert a larger number of IMD cases than the current schedule (range, 189 to 256 averted IMD cases, depending on schedule). The larger number of averted total IMD cases compared with the current vaccination schedule is primarily driven by a larger number of prevented serotype B cases (range, 55 to 111 serotype B cases). Of all base-case vaccination scenarios evaluated, disease prevention would be greatest with two doses of MenABCWY vaccine administered at age 11 years and one dose at age 16 years (scenario 10, 256 averted cases [111 serotype B, 146 serotypes A, C, W, and Y]). 3.2 Sensitivity analysis based on alternative vaccination rates If MenABCWY vaccination coverage at age 16 years were to increase to the level observed with HPV vaccines at 11-12 years of age (68.1%), the greatest impact on meningococcal disease prevention would be provided by two doses of MenABCWY vaccine at age 11 years and one dose at age 16 years (scenario 11, 299 total cases averted). This schedule would also prevent the greatest number and percentage of serotype B cases among all preventable cases over a 10-year period (140 averted serotype B cases [46.8%]). The next-best regimen was one dose of MenABCWY vaccine at age 11 years and two doses of MenABCWY vaccine at age 16 years (scenario 5, 263 averted cases, 103 serotype B, and 159 serotypes A, C, W, and Y). Furthermore, a similar number of cases would be averted with or without the currently recommended inclusion of the MenACWY dose at age 11 years (Scenario 2 [234 averted cases, 74 serotype B, 159 serotypes A, C, W, and Y] and 8 [220 averted cases, 74 serotype B, 146 serotypes A, C, W, and Y]). In contrast, a regimen assuming MenABCWY vaccination coverage equal to the current two-dose MenB vaccination schedule at age 16 years (17.2% with one or more doses) would have resulted in far fewer estimated IMD-prevented cases, primarily due to fewer averted serotype B cases (Scenarios 3, 6, 9, and 12). Under this vaccination coverage assumption, the smallest number of total IMD cases averted would result from a two-dose MenABCWY vaccine regimen at age 16 years (scenario 9, 93 total cases averted [19 serotype B, 74 serotypes A, C, W, and Y]). 4.0 Discussion To our knowledge, this is the first study to model the impact of the pentavalent MenABCWY vaccine in protecting against meningococcal disease caused by the five most prevalent disease-causing serotypes (i.e., serotypes A, B, C, W, and Y) in the context of the U.S. adolescent meningococcal immunization platform. Globally, various monovalent, bivalent, or quadrivalent meningococcal vaccine formulations targeting various combinations of these five serotypes are used to help protect against meningococcal disease. In several countries, ongoing surveillance efforts have detected changes in circulating disease-causing meningococcal serotypes. These epidemiological shifts have prompted changes in some national vaccination strategies to include the MenABCWY vaccine for comprehensive protection against IMD. Deployment of the MenABCWY vaccine would potentially protect against 94% of the estimated IMD cases caused by these five serotypes worldwide.

[0315] In the United States, MenACWY vaccination for adolescents has been recommended since 2005, and MenB vaccination recommendations were issued in 2015. By eliminating the need for multiple injections of two different vaccines at different ages, a single vaccine could simplify immunization schedules, potentially improving vaccination coverage and enhancing protection against the most prevalent disease-causing serotypes. Based on the current schedule and vaccination rates of the MenACWY and MenB vaccines in the United States, our model estimates that vaccination with both the MenACWY and MenB vaccines could potentially avert 165 cases of IMD over a 10-year period compared with no vaccination. Assuming MenABCWY vaccination rates are similar to the current MenACWY vaccination rates noted in the 2018 CDC NIS-Teen survey, our model estimates that replacing one or more doses of MenACWY or MenB vaccine with the MenABCWY vaccine could result in as many as 256 averted cases of IMD among U.S. adolescents while simultaneously reducing the number of recommended vaccine injections. Indeed, most of the scenarios examined in this study demonstrate the added benefit of a single MenABCWY vaccine in terms of a greater number of averted cases compared with the current schedule.

[0316] Immunization delivery to adolescents is challenging, in part because the rate of preventive health visits where immunization typically occurs steadily declines after age 16, and this, combined with a shift from pediatricians to healthcare providers who are typically less involved in adolescent immunization, may contribute to suboptimal protection against disease in this age group. An age-based platform for MenABCWY vaccination would support and catalyze adolescent immunization by allowing vaccine administration at an age when adolescents are more likely to receive and respond to multi-dose regimens, reducing the number of injections and visits. Importantly, immunization of adolescents with both the MenACWY conjugate vaccine and the recombinant protein MenB vaccine elicits protective immune responses after primary vaccination and robust responses after booster doses. Together, these data support not only the existing MenACWY and MenB immunization platform for US adolescents, but also flexible MenABCWY vaccination schedules that would allow adolescents to begin the vaccination series anywhere between 11 and 16 years of age and maintain protection throughout their highest-risk period.

[0317] Studies have shown that socioeconomic status, education, and race also play a role in vaccination awareness, access, and utilization, as well as series completion rates. Therefore, the MenABCWY vaccine may help reduce these disparities by simplifying meningococcal vaccination recommendations, thereby reducing vaccine access issues and eliminating confusion surrounding existing MenB and MenACWY vaccine recommendations. Furthermore, combination vaccines have generally been shown to improve vaccination rates across various age groups. [Example]

[0318] Breadth of human immune responses to TRUMENBA: A summary of fHBP variants expressed by MenB strains susceptible in hSBA Introduction and Objectives: TRUMENBA (bivalent rLP2086), a vaccine for preventing meningococcal serogroup B (MenB) disease, contains two protein antigens, variants of the meningococcal factor H-binding protein (fHBP). fHBP exists as two subfamilies, A and B. Within each subfamily, hundreds of unique fHBP variants have been identified. Despite this sequence diversity, a vaccine containing one protein from each subfamily was demonstrated to induce broad coverage across MenB strains, representing the diversity of fHBP variants. Licensing was based on the vaccine's ability to elicit antibodies that initiate complement-mediated killing of invasive MenB strains in a serum bactericidal assay (hSBA) using human complement. Due to the endemic nature of meningococcal disease, it is not possible to predict which fHBP variants an individual may be exposed to. Therefore, TRUMENBA (bivalent rLP2086) ... Continuing to explore the coverage afforded by ENBA, further evidence illustrating the breadth of immune coverage is presented herein.

[0319] Materials and Methods: MenB invasive strains (n ​​= 109) were selected to confirm the breadth of coverage of TRUMENBA. Strains encoded 22 and 16 unique subfamily A and subfamily B fHBP variants, respectively. Expression of fHBP on the bacterial surface was determined using a flow cytometric meningococcal antigen surface expression (MEASURE) assay. Investigational hSBA was performed using matched pre- and post-vaccination sera from young adults. Strains were considered susceptible to TRUMENBA-immune sera if a four-fold increase in hSBA titer was achieved between pre- and post-vaccination serum samples.

[0320] Results: Of 109 strains, 87 (nearly 80%) were sensitive to TRUMENBA immune serum in hSBAs. This included strains expressing fHBP mutants A02, A28, A42, A63, A76, B05, B07, B08, B13, B52, and B107, in addition to previously reported mutants. The majority of strains that could not be killed had fHBP expression levels below the level considered sufficient to initiate bactericidal killing in hSBAs. See Figure 3, Tables 1, 2, and 3.

[0321] [Table 20]

[0322] Table 21 fHBP variants expressed by MenB strains killed by TRUMENBA-immune serum in hSBAs (% amino acid sequence identity with A05 (SEQ ID NO: 1) and B01 (SEQ ID NO: 2))

[0323] [Table 21]

[0324] Table 22 fHBP variants expressed by strains with MFI less than 1000 and not killed by TRUMENBA immune serum in hSBA * (% amino acid sequence identity with vaccine antigens A05 (SEQ ID NO: 1) and B01 (SEQ ID NO: 2))

[0325] [Table 22]

[0326] Conclusions: hSBA is recognized as a surrogate for meningococcal vaccine efficacy. Assay complexity precludes demonstration of bactericidal activity of TRUMENBA-immune sera against MenB strains expressing each of the hundreds of unique fHBP sequence variants. To illustrate the breadth of immune coverage conferred by TRUMENBA, we show that MenB strains expressing additional diverse fHBP variants can be killed in hSBA despite being non-homologous to the vaccine antigen. [Example]

[0327] Selection of diverse strains to evaluate broad coverage of bivalent FHbp meningococcal B vaccines Meningococcal infection typically results in asymptomatic colonization of the upper respiratory tract, but in some individuals, bacteremia and invasive meningococcal disease (IMD) occur. IMD commonly presents as meningitis and / or sepsis, with pneumonia, septic arthritis, epiglottitis, and otitis media observed less frequently. A high mortality rate is associated with IMD (10%–15%), and approximately 20% of survivors suffer from severe lifelong sequelae, including limb amputation, hearing loss, and neurological impairment.

[0328] Nearly all meningococcal disease worldwide is caused by six of the 12 characterized meningococcal serotypes (i.e., A, B, C, W, X, and Y). Effective capsular polysaccharide-based vaccines have been developed for serotypes A, C, W, and Y. However, due to its similarity to polysialic acid structures present on human neuronal cells, the MenB polysaccharide is poorly immunogenic. In recent years, meningococcal serotype B (MenB), in particular, has been associated with the majority of IMD in Europe, the United States, Canada, Australia, and New Zealand. Outer membrane vesicle (OMV)-based vaccines have been successfully used to control epidemics caused by a single MenB outbreak strain, but the resulting immune response is primarily directed against the highly variable porin A protein (PorA). Therefore, efficacy is generally limited to the target strain. Consequently, surface-exposed proteins capable of eliciting protective bactericidal antibodies across diverse MenB strains are being explored for the development of broadly effective MenB vaccines.

[0329] Factor H-binding protein (FHbp, also known as LP2086 and GNA1870), a conserved surface-exposed lipoprotein expressed on nearly all strains of MenB, was identified as such a target. Based on amino acid sequence, FHbp variants were separated into two immunologically distinct subfamilies (termed subfamily A and subfamily B), with each MenB strain expressing a single subfamily variant (see Fig. 1A).

[0330] MenB-FHbp (TRUMENBA®, bivalent rLP2086, Pfiz MenB-4C (Bexsero®, 4CMenB, GlaxoSmithKline Vaccines, Srl, Siena, Italy) is a bivalent recombinant protein MenB vaccine composed of equal amounts of two recombinant lipidated FHbp antigens, one from subfamily A (variant A05) and the other from subfamily B (variant B01). Importantly, this combination of FHbp variants is predicted to provide protection against diverse MenB strains. MenB-FHbp has been licensed for the prevention of IMD in several countries and regions, including the United States, Canada, Europe, and Australia. Another MenB vaccine, MenB-4C (Bexsero®, 4CMenB, GlaxoSmithKline Vaccines, Srl, Siena, Italy), also contains a recombinant FHbp component (non-lipidated variant 1.1 from subfamily B) as well as two other recombinant protein antigens and OMVs. Thus, MenB-4C differs from MenB-FHbp, which contains two variants of a single antigen to provide broad coverage.

[0331] The human complement serum bactericidal assay (hSBA) measures complement-dependent, antibody-mediated lysis of meningococcal bacteria. The hSBA titer is defined as the highest serum dilution that kills 50% or more of the assay bacteria. A hSBA titer of ≥1:4 correlates of acceptable protection against meningococcal disease, and the hSBA response rate, based on this correlation, has been used as a surrogate for meningococcal vaccine efficacy. SBA response rates have been specifically correlated with the natural protection of serotype C and A polysaccharide vaccines. Because serotype-specific polysaccharides are not variable, a single strain from each serotype was sufficient to infer broad vaccine coverage. MenB OMV vaccines have also been effective, and vaccine-induced hSBA titers correlated with protection against outbreak-causing target strains. Given the variability in protein sequence and expression levels among different meningococcal disease strains, accurately predicting strain coverage for protein-based vaccines using hSBA is more complex than for vaccines targeting capsular polysaccharides. For example, PorA (PorA) is the primary target of serum bactericidal antibodies and confers protection after immunization with OMV vaccines. PorA is a cell surface porin with a small, exposed cell surface region with a high degree of sequence diversity. It has been estimated that to protect against approximately 80% of strains causing sporadic MenB disease in the United States, protective immunity would need to be demonstrated with strains expressing 20 different PorA serovars. Historically, OMV vaccines have contained a single PorA and have not demonstrated protection against strains with PorA sequences that are nonhomologous in amino acid sequence compared to the vaccine antigen. Therefore, the selection of representative test strains to demonstrate that vaccine-induced antibodies can be effective against meningococcal disease strains is paramount for protein-based vaccines.

[0332] In preclinical and early clinical studies, immune sera elicited by MenB-FHbp demonstrated broad bactericidal antibodies capable of killing diverse MenB strains containing FHbp subfamily A and B variants non-homologous to the vaccine FHbp variants A05 and B01. In an initial assessment of the potential breadth of MenB-FHbp coverage, 100 MenB isolates representing diverse FHbp variants, geographic origins, and genetic backgrounds were tested in hSBAs using MenB-FHbp-immune rabbit sera. Of the 100 strains tested, 87 were killed in these hSBAs. Analysis of the 13 strains that were not killed suggested that a threshold FHbp surface expression level on a given MenB strain influenced the hSBA response. We then determined the threshold FHbp surface expression level above which an isolate was predicted to be killed in hSBAs. Further investigation of potential factors determining strain susceptibility found that killing was largely independent of FHbp sequence variant, multilocus sequence type, or PorA subtype.

[0333] To select strains with broad antigenic and epidemiological diversity for clinical trials, over 1200 invasive MenB disease isolates were collected from laboratories and health institutions in the United States and Europe. The four primary MenB test strains were collected to represent the prevalence of MenB isolates present at the time of collection. All strains contained the FHbp gene. An unbiased approach was used to select four antigenically and epidemiologically diverse representative test strains for use in MenB-FHbp immunogenicity studies. Selection criteria included expression of FHbp variants nonhomologous to vaccine antigens and adequately reflecting the diversity of FHbp in MenB disease isolates, low to moderate FHbp surface expression levels, and low baseline hSBA seropositivity. These four primary MenB test strains express FHbp variants from both FHbp subfamilies (strains [variants]: PMB2001 [A22], PMB80 [A56], PMB2707 [B24], and PMB2948 [B44]; see Figure 1A).

[0334] To complement the immunogenicity data generated using the four primary MenB test strains and demonstrate that the immune responses to the four primary MenB test strains are predictive of the immune response to the diversity of FHbp variants expressed by MenB disease-causing isolates, we developed an hSBA using 10 additional test strains. The 10 additional test strains were selected to include prevalent FHbp variants found among MenB disease-causing strains in the United States and Europe. Here, we (i) describe the strategy and criteria used to select the 10 additional test strains and (ii) demonstrate that the immune responses measured by hSBA using the four primary MenB strains are predictive of responses obtained using the 10 additional test strains, presenting data further demonstrating and supporting the broad coverage of immune responses elicited by MenB-FHbp. result Sources and selection criteria for additional MenB test strains Nine of the 10 additional MenB test strains were obtained from a collection of 1,263 invasive disease-causing MenB strains (MenB isolate collection). The MenB isolate collection included US strains from Active Bacterial Core Surveillance sites (2000–2005), covering approximately 13% of the population. The European isolates (2001–2006) were from public health laboratories in Norway, France, and the Czech Republic, as well as the Manchester Health Protection Agency (covering England, Wales, and Northern Ireland). They were systematically collected (including every seventh or eighth isolate received in the order received by the national reference laboratory) and represented approximately 13% of invasive MenB isolates during the period. The strain expressing FHbp variant A07 was obtained from an expansion of the MenB isolate collection to include an additional 551 disease-causing MenB strains from Spain and Germany (n = 1,814). The expanded MenB isolate collection used as the A07 expressing strain in the MenB isolate collection was not suitable due to the low surface expression of FHbp on these strains, high baseline seropositivity, and lack of a readily available source of complement.

[0335] The criteria used to select additional MenB test strains were: (i) FHbp variant prevalence among strains causing MenB disease in the United States and / or Europe; (ii) FHbp variants required to differ from those expressed by MenB primary test strains; (iii) in vitro FHbp expression levels below the median level of the respective FHbp variant group to ensure the strain is representative of the variant group to which it belongs; (iv) technical compatibility in hSBA; and (v) the ability to be considered as the major clonal complex of the variant group (if a major complex exists). Strains meeting these criteria also required technical compatibility with hSBA, including the availability of sufficient lots of appropriate human complement (Figure 2). Strains within each FHbp variant group with expression levels below the cutoff level (i.e., below the median level of the respective FHbp variant group) were randomly selected, and the first strain within the FHbp variant group that met the required genetic, phenotypic, and hSBA development criteria became the additional MenB test strain. An exception to this methodology was made in collaboration with the US FDA and using the guidance provided, based on its previous use in a Phase 2 study. This was performed on a strain expressing the FHbp mutant B03 selected by the method described above. Characterization of additional MenB test strains Ten additional selected MenB test strains express FHbp variants A06, A07, A12, A15, A19, A29, B03, B09, B15, and B16, which are distinct from those in the four primary test strains (A22, A56, B24, and B44) and have distinct sequences compared to the vaccine antigen (Table 23). The specific variants expressed by the four primary test strains are present in 42.0% (530 / 1263) of the disease-causing isolates in the MenB isolate collection, and the specific variants expressed by the 10 additional test strains are present in an additional, non-overlapping 38.8% (490 / 1263) of the disease-causing isolates in the MenB isolate collection (Figure 1B).

[0336] [Table 23]

[0337] Immunogenicity analysis: subjects with hSBA titers ≥ LLOQ for 10 additional strains Four primary strains were used to evaluate serologic responses after two or three doses of MenB-FHbp in subjects participating in two pivotal phase 3 studies in adolescents and young adults. Serologic responses to 10 additional hSBA strains were evaluated in a subset of study subjects. The majority of subjects had hSBA ≥ lower limit of quantification (LLOQ, i.e., hSBA titers equal to 1:8 or 1:16, depending on the strain) 1 month after dose 2 and 1 month after dose 3 for the primary (64.0%-99.1% and 87.1%-99.5%, respectively) and each of the 10 additional MenB test strains (51.6%-100.0% and 71.3%-99.3%, respectively) (Table 24). For the primary and additional MenB test strains, a substantial increase from baseline in the proportion of subjects achieving hSBA titers ≥ LLOQ was observed in MenB-FHbp recipients (0-, 2-, and 6-month schedules) after the second MenB-FHbp dose, with a further increase observed after the third dose.

[0338] [Table 24-1]

[0339] [Table 24-2]

[0340] Positive predictive values ​​of primary and additional strains The relationship between vaccine-induced hSBA responses between the primary MenB test strain and 10 additional MenB test strains was evaluated (Table 25). Within the FHbp subfamily, the positive predictive value (PPV) was greater than 80% for most primary / additional strain pairs 1 month after dose 3. Thus, immune responses measured by hSBA using the primary test strain were highly predictive of immune responses to additional strains within the same subfamily. PPVs 1 month after dose 2 were typically slightly lower than those observed 1 month after dose 3 and ranged from 61.6% to 100% and 70.0% to 100% for subfamily A and B strain pairs, respectively, across the study. In summary, all PPVs demonstrated high predictability for protective responses compared with hSBA responses of the primary and additional strains.

[0341] [Table 25-1]

[0342] [Table 25-2]

[0343] Consideration A critical component of the clinical evaluation of the MenB-FHbp vaccine to determine breadth of protection was the development of hSBAs using test strains with surface protein antigens whose sequence and expression variability is representative of the diversity of MenB disease-causing strains present at the time of collection. As described in a Phase 3 study in adolescents and young adults, hSBA response data for four primary MenB test strains, all expressing FHbp variants nonhomologous to the vaccine antigens, strongly suggest that the bivalent MenB-FHbp vaccine provides broad coverage across diverse disease-causing meningococcal strains. 10 The additional MenB test strains provide supportive immunological data for MenB-FHbp and further validate the use of the four primary test strains to measure immune responses to MenB-FHbp. Because the responses obtained with the four primary test strains are predictive of those obtained with the additional 10 test strains, the immunological responses obtained by evaluating the primary strains in the hSBA are representative of the diversity of strains causing invasive MenB disease.

[0344] For hypothesis-driven immunogenicity evaluation of MenB-FHbp in licensing studies, an unbiased approach was used to select four primary MenB test strains from a panel of disease-causing MenB strains collected in the United States and Europe. Ten additional MenB hSBA test strains were selected using a similar approach, considering specific selection criteria to ensure that the test strains were representative of the antigenic diversity of MenB isolates. Combined, the 14 MenB test strains represent the majority of circulating meningococcal FHbp, with FHbp variants representing approximately 80% of circulating invasive disease-causing isolates in the United States and Europe.

[0345] Positive predictive value analyses were used to determine the association of immune responses measured by hSBA between primary and additional test strains expressing FHbp within the same subfamily. All PPV analyses demonstrated a high predictability of protective responses to the primary strain for protective responses observed against additional strains. These PPV analyses indicate that the responses observed against the four primary MenB test strains are representative of responses to other disease-causing MenB strains that express additional sequence-diverse FHbp variants distinct from the vaccine antigen variants.

[0346] MenB-FHbp-induced responses, as measured by hSBA, to four primary and 10 additional MenB test strains were assessed using sera from individual vaccine recipients. By determining the proportion of vaccinated subjects with functional bactericidal antibodies, an assessment of the breadth of MenB-FHbp coverage at the individual level was determined, which is not possible with pooled sera. The four primary MenB test strains were selected to represent the diversity of MenB disease-causing IMDs and thus support the potential breadth of MenB-FHbp coverage using hSBA. The response of individuals with hSBA titers ≥ 1:4 is an acceptable correlate of protection and a surrogate for meningococcal vaccine efficacy. Thus, responses provide a comprehensive and biologically predictive assessment of the breadth of vaccine coverage. The relevance of the hSBA responses to the four primary MenB test strains to explain the range in vaccination rates is supported by the demonstration of protective bactericidal responses by MenB-FHbp observed against diverse and contemporaneous MenB outbreak strains from Europe and the United States, as well as against strains that do not cause MenB disease (i.e., meningococcal serotypes C, Y, W, and X).

[0347] Another method, the enzyme-linked immunosorbent assay-based meningococcal antigen typing system (MATS), has been used to predict MenB-4C vaccination coverage. However, MATS only predicts coverage of antigens specific to MenB-4C and is not useful for assessing coverage of other vaccines with different antigen compositions. Specifically, MATS measures antigen expression, not bactericidal activity, and is reported as relative potency compared to a reference strain for each antigen. A strain is considered susceptible to killing if the relative potency of any one of the component antigens is commensurate with the bactericidal activity of MenB-4C-immune serum (i.e., achieves a positive bactericidal threshold). However, because MATS does not use serum from vaccinated individuals, the assay cannot predict the proportion of a population that will achieve an hSBA titer of ≥ 1:4 (i.e., a correlation of protection) in response to immunization.

[0348] It is worth noting that there are limitations to conducting hSBA. For example, hSBA is labor-intensive and may require large amounts of serum and assay-compatible complement, especially when evaluating a larger number of strains and / or sera. Furthermore, interlaboratory differences in the performance of assay reagents and strains used in hSBA limit the comparison of responses between vaccines and the assessment of breadth of coverage. A known limitation of PPV analysis is the dependence of the magnitude of response on prevalence (i.e., in this setting, the proportion of subjects achieving hSBA ≥ LLOQ for the additional strains). However, it is noteworthy that in this analysis, although there was a range of postvaccination responses to the additional strains (1 month after doses 2 and 3), the PPV was uniformly high.

[0349] Taken together, immunogenicity data from 10 additional MenB hSBA test strains support response data from the four primary MenB hSBA test strains and confirm the broad coverage of MenB isolates conferred by MenB-FHbp. This is the first study to apply MenB strain epidemiology, in conjunction with recognized surrogates of protection (hSBA) for vaccine antigen sequence and expression, to rigorously evaluate the elicited immune response of a MenB vaccine and use this knowledge to lead to vaccine licensure. method Quantification of FHbp surface expression For all strains, FHbp surface expression was quantified by the MEASURE assay, a flow cytometry assay using a monoclonal antibody (MN86-994-11) recognizing a conserved FHbp epitope common to both FHbp subfamilies. Details of the MEASURE assay have been previously described. The cutoff level adopted for each FHbp mutant group was the observed median mean fluorescence intensity plus one standard deviation, using a precision estimate of 25.2% relative standard deviation. Immunogenicity analysis Each of the 10 additional MenB test strains was used in an hSBA against test sera from subjects participating in two pivotal phase 3 studies of MenB-FHbp. A total of 900 subjects from each study were divided into three subsets (n = 300 each), and the 10 additional test strains were distributed across these subsets, with two subsets containing three test strains each and one subset containing four test strains. To ensure that each study yielded at least 150 evaluable hSBA results, the subsets included samples from 300 subjects. Immune responses measured by hSBA using phase 3 clinical study sera were based on an assay LLOQ, which was an hSBA titer equivalent to 1:8 or 1:16, depending on the strain. Positive predictive value analysis The PPV of each primary / additional strain pair within an FHbp subfamily was defined as the proportion of subjects responding to the additional strain (hSBA titer ≥ LLOQ for the additional strain) among the total number of primary strain responders (hSBA titer ≥ LLOQ for the primary strain). PPV analysis assessed whether the observed hSBA responses to the four primary strains predicted immune responses to additional strains expressing FHbp from the same subfamily. [Example]

[0350] The pentavalent meningococcal (MenABCWY) vaccine is safe and well tolerated, with non-inferior immunogenicity to co-administered MenB-FHbp and MenACWY-CRM in a phase 2 study in healthy adolescents and young adults BACKGROUND: Meningococcal serotypes A, B, C, W, and Y cause nearly all meningococcal disease worldwide. Vaccination is complicated by different dosing recommendations for serotype B (MenB) and quadrivalent (MenACWY) vaccines, which could be resolved with a single pentavalent vaccine. This study in adolescents and young adults compared two licensed vaccines, MenB-FHbp (TRUMENBA®, bivalent rLP2086) and A new pentavalent MenABCWY vaccine combining MenACWY-TT (NIMENRIX®) and MenACWY-TT into a single vaccine was evaluated.

[0351] Methods: In this ongoing, randomized, controlled, observer-blinded, multicenter study (NCT03135834), healthy MenB vaccine-naive and MenACWY-naive or -experienced individuals aged 10-25 years were randomized 1:2 to receive MenABCWY (0 and 6 months) or MenB-FHbp (0 and 6 months) and MenACWY-CRM (0 month). Immune responses were measured by human complement-based serum bactericidal assay (hSBA) against serogroups A, C, W, and Y strains and four diverse vaccine-heterologous MenB strains. Endpoints included the percentage of subjects achieving a 4-fold or greater increase in titer from baseline. Noninferiority of immune responses was assessed a priori with a 10% margin (lower limit of 95% CI >-10%). Safety was assessed.

[0352] Results: Following dose 2, a high percentage of MenABCWY (n = 543) and MenB-FHbp (n = 1057) recipients achieved a 4-fold or greater rise in titers against each of the four MenB strains (75.8-94.7% vs. 67.4-95.0%) and at least the lower limit of quantification for all four strains combined (79.9% vs. 74.3%, Figure 1A). MenABCWY was noninferior to MenB-FHbp for all five endpoints. MenABCWY was also noninferior to a single MenACWY-CRM dose; depending on prior MenACWY experience, 75.5-96.9% and 93.0-97.4% of MenABCWY recipients achieved a 4-fold or greater rise in titers against serogroups A, C, W, and Y after doses 1 or 2, respectively (Figure 1B). Local reactions and systemic events after MenABCWY or MenB-FHbp were similarly frequent, most were mild / moderate in severity ( Figure 2 ), and were not affected by MenACWY experience. 6.2.2.3 Immunogenicity Results from the MenABCWY Clinical Development Program (Study B1971057) This portion of the study was the Phase 2 component of the overall study. For the MenB component of MenABCWY, the percentages of MenABCWY and bivalent rLP2086 + MenACWY-CRM recipients achieving a 4-fold increase from baseline in hSBA titers 1 month after vaccination 2 were 75.8% and 73.8%, respectively, for PMB80 (A22), 94.7% and 95.0%, respectively, for PMB2001 (A56), 76.1% and 67.4%, respectively, for PMB2948 (B24), and 91.7% and 86.4%, respectively, for PMB2707 (B44). The proportion of MenABCWY and bivalent rLP2086+MenACWY-CRM recipients achieving a combined response (hSBA titers ≥ LLOQ for all four MenB test strains) 1 month after vaccination 2 was 79.9% and 74.3%, respectively.

[0353] Conclusions: These results demonstrate that, despite previous ACWY experience, MenABCWY, whether given as a single dose or as a two-dose series spaced 6 months apart, provides a high degree of protective immunity against MenB after two doses and against MenACWY after one or two doses, similar to that achieved when bivalent rLP2086 (0, 6 months) and MenACWY-CRM (0 months) are administered separately. The 4-fold immune response from baseline with MenABCWY was robust and noninferior to MenB-FHbp and MenACWY-CRM administered separately. Vaccination on a 0, 6-month schedule is safe and well tolerated. The favorable benefit / risk profile supports further development of MenABCWY as a simplified alternative to current meningococcal vaccination practices.

[0354] Data from Study B1971057 (MenABCWY [FIH]) demonstrated that when administered to healthy individuals aged 10 to 25 years, the response to MenABCWY was significantly higher than that of TRUMENBA (Meningococcal B Bivalent Recombinant Lipoprotein 2086 Vaccine) (MenB Evaluation). and MENVEO (Meningococcal (A, C, Y, and W-135) Oligosaccharide CRM197 Conjugate Vaccine) (MenACWYCRM Evaluation) (see Section 5.2). No immune interference between the component parts of MenABCWY was observed in this FIH study. [Example]

[0355] Effect of transport stress on suspension vaccines This example illustrates the effects of transportation stresses (shock / drop, vibration, low pressure / high altitude, and temperature) on suspension vaccines. Redispersion of vaccine suspensions is an important consideration. Understanding the factors that may affect redispersion time is a key product development objective in order to minimize redispersion time for the end user. We present a systematic method to evaluate the parameters that affect redispersion time of suspension vaccines and associated control strategies.

[0356] Suspension vaccines are thermodynamically unstable systems that present significant challenges in maintaining physical properties during storage and transportation compared to liquids or lyophilized powders. While transportation stress can affect product quality and the physical properties of vaccine products, this example focuses on the effect of transportation stress on the physical properties of suspension vaccines. Implications for product quality are out of scope, as product quality is molecule-dependent and can be assessed by product-specific analytical and biological methods.

[0357] Aluminum-containing adjuvants have been included in vaccines to enhance or modify immune responses to antigens for over 70 years. Aluminum-containing vaccines are suspensions whose internal phase consists of an insoluble aluminum-containing salt (aluminum phosphate or aluminum hydroxide) and whose external phase is a liquid vehicle. Adding an insoluble adjuvant to a vaccine drug product creates a suspension in which the dispersed phase tends to settle over time. Ideally, the suspension should be homogeneous, and any settling that occurs during storage can be easily redispersed upon agitation. Prefilled syringes containing vaccine suspensions are compared before and after redispersion. Redispersion problems are often common after prefilled syringes are filled with suspensions and stored for extended periods. Numerous studies have focused on adding controlled flocculants or polymeric additives to form large, loose, and easily dispersible precipitates. Some commercially available aluminum-based vaccines have clear package insert instructions to "shake vigorously immediately before use." On April 26, 2010, WHO recommended the recall and destruction of all lots of SHAN 5 vaccine as a precautionary measure after an incident in which SHAN 5 vaccine vials contained a white precipitate that was difficult or impossible to resuspend. Therefore, redispersion of vaccine suspensions should be thoroughly evaluated to understand factors affecting redispersion times, so that mitigation measures can be implemented, if necessary.

[0358] Interactions between particle size, charge, and settling rate. In exploring the causes of increased redispersion time, it is essential to understand the factors that affect suspension stability. In this section, a background to suspension stability considerations is presented along with case studies of two suspensions to understand the interplay between suspension particle size, charge, and settling.

[0359] In an ideal suspension system, the dispersed phase remains suspended for an extended period of time, and if settling of the sparse system occurs, the dispersion can be easily resuspended or dispersed. Suspension stability can be achieved by thermodynamic or kinetic means. Thermodynamic stabilization techniques involve adding charges to the suspension particle surfaces, thereby inducing steric hindrance due to interparticle repulsion, resulting in suspension stabilization. On the other hand, kinetic techniques involve increasing the viscosity of the suspension, resulting in reduced settling of the suspension, thus providing stability. Particle size is affected by thermodynamic It plays an essential role in this interplay between mechanical and kinetic stability. In suspension systems with particle sizes larger than submicron, the density difference between the transparent and dispersed phases is a significant factor, which can lead to the settling of the dispersed phase due to gravity. It has been reported that particle size can affect the flocculation or coagulation of suspension systems. For example, PEG is added to lots of solid / liquid suspensions for injection to allow solid active ingredient particles to form larger flocculants, thus making the precipitate easier to resuspend. While supermicron particles are ideally suited to slow the kinetics of dissolution, they are much more affected by the effects of gravity, which can result in a somewhat compact precipitate that is hardly resuspended.

[0360] Sedimentation or settling is the result of collective interactions in concentrated suspensions. Collective interactions include hydrodynamics and interparticle interactions (determined by charge-charge interactions). Hydrodynamic effects cause backflow retardation, which is the backflow of fluid to counteract the motion of settling particles. Interparticle interactions are due to attractive self-depletion and repulsive structural forces in concentrated dispersions.

[0361] To illustrate the interplay between charge, sedimentation rate, and particle size, two suspension systems are illustrated. The vaccine suspension drug product under investigation is designated Suspensions 1 and 2 in this illustration. Suspensions 1 and 2 exhibit comparable particle sizes (approximately 15 μm). These suspensions behave very differently when comparing sedimentation rates. Suspension 1 settles significantly faster, with a sedimentation rate of approximately 0.04 abs / min, while Suspension 2 takes several weeks to even begin to precipitate. This difference is evident despite having comparable particle size distributions. As previously discussed, understanding the charge on the particle surface is essential. Suspension 1 has a zeta potential of approximately -5 mV, while Suspension 2 has a zeta potential of -45 mV. Because Suspension 1 has only a small charge on the particle surface, thermodynamic suspension instability promotes interparticle attraction and ultimately precipitation of the dispersed phase.

[0362] On the other hand, the high charge of the system in Suspension 2 makes it thermodynamically stable, and interparticle repulsion leads to significantly slower precipitation of the dispersed phase. Based on this, Suspension 2 offers the greatest thermodynamic stability and is desirable. The main question is whether a thermodynamically stable suspension is easier to disperse after settling, or vice versa. How does this translate to ease of suspension in a syringe for a vaccine suspension? Our data using these two suspensions suggests that Suspension 1, while thermodynamically unstable due to the lack of charge, leads to interparticle interactions and potentially the formation of larger flocculants, resulting in faster settling and therefore easier resuspension of the dispersed phase system. On the other hand, Suspension 2 takes significantly longer to settle due to the charge-charge polarity, but once settled, the finer particles are tightly packed, forming a settled dispersed phase that is significantly more difficult to resuspend.

[0363] Based on these observations and background, two main questions were tested in our study to understand the increased redispersion time of Suspension 1 vaccine: Could transportation stress alter the thermodynamic suspension stability, resulting in increased redispersion time? What is the effect of individual transportation stress components during transportation, and is there a correlation with increased redispersion time? Effect of transport stress on thermodynamic suspension stability. In another study, vaccine drug product suspensions were subjected to simulated shipping stresses, including shipping temperature, shock / drop, and vibration. Thermodynamic suspension stability was determined by measuring charge (zeta potential), particle size distribution, and sedimentation rate before and after shipping.

[0364] The drug product suspension exhibits a zeta potential value of approximately -5 mV, which indicates that there is a slight This suggests the presence of a charge. This thermodynamic instability of the suspension promotes interparticle attraction and ultimately precipitation of the dispersed phase. Our data suggest that there were no obvious changes in the zeta potential values, particle size distribution, and measured precipitation rates before and after the simulated syringe transport. These results suggest that transport stress does not alter the thermodynamic suspension stability of the drug product suspension. Because these properties do not change after transport, it can be concluded that thermodynamic stability is not the driving factor behind the observed increase in redispersion time, which entails the need to evaluate the effect of transport stress and understand whether any specific physical mechanisms contribute to the observed redispersion time. Effect of individual transport stresses on redispersion time When transporting a package, the product undoubtedly experiences a variety of transport stresses. These include shock / drop stress as the package is dropped from various heights, and vibration during transport by either truck or airplane (although the amplitude of the vibration frequency differs between airplane and truck). The effect of each transport stress on redispersion time was evaluated when drug product syringes were subjected to simulated shipping. 11The study design also included an evaluation of the effects of shipping stress as a function of syringe orientation. Syringes were stored in three separate orientations: tip cap down, tip cap up, and tip cap horizontal. The study aimed to decipher the individual stresses and understand their effects.

[0365] It was observed that shock / drop stress reduced the redispersion time in the tip cap down direction, while the effect of shock / drop on redispersion was negligible in the tip cap horizontal and tip cap up directions.

[0366] On the other hand, aircraft vibration increased the redispersion time in the tip cap down direction. This is an interesting finding and may be related to the vibration frequency experienced during aircraft transport. The vibration frequency when the syringe is in the tip cap down direction may further encourage suspension particles to move down the inner diameter of the syringe. The effect of aircraft vibration was negligible for syringes placed on the tip cap and horizontally. Similar to aircraft vibration, truck vibration increased the redispersion time in the tip cap down direction. These findings are similar to aircraft vibration, but the magnitude of the increase in redispersion time was greater than in the case of aircraft vibration. Furthermore, consistent with aircraft vibration, the effect of truck vibration was negligible for syringes placed on the tip cap and horizontally.

[0367] Also, when syringes were evaluated for redispersion time for combined stresses (shock / drop, airplane vibration, truck vibration shock / drop), the data were confounded, suggesting that in real-world shipping, perhaps one or more modes of shipping stress dominate the results and may produce entirely different results.

[0368] Based on the differences seen with aircraft and truck vibrations, it was interesting to note that the intensity of the vibration frequency was a significant factor. The increased redispersion time when the syringe was placed tip-cap downwards may be due to gravity, where the increased downward movement of suspension particles due to vibration encourages larger particles to pack tightly together and smaller particles to fill the vacant spaces. Particles at the bottom, especially those near the inner diameter of the syringe, are gradually pushed down by the gravity of those above.

[0369] When the drug product is delivered in the tip-cap-up orientation, the effect of gravity is still present, but settling occurs on the wider surface of the plunger rather than the narrow bore. Clot packing is not evident. When the drug product is delivered in the tip-cap-horizontal orientation, lateral motion resulting from Brownian motion and convective currents from vibration overcome gravity. Even if settling does occur, it occurs over the broad surface of the syringe wall, and the probability of particle packing is small. Therefore, resuspension is not an issue in either the tip-cap-up or tip-cap-horizontal orientation, and the tip-cap-horizontal orientation is not evident. The horizontal top cap orientation gives slightly better results, probably due to the larger surface area. Upon transport, transport vibration is the dominant force that leads to the final state of compaction of the sediment. Depending on the syringe orientation and the different surface areas available, the redispersion time scales with these open surface areas. Mitigation strategies to reduce high redispersion times Based on the transport simulation study data, it is clear that either tip cap horizontal or tip cap up orientation can mitigate the higher redispersion time of the vaccine suspension. Effect of transport temperature on suspension vaccines In addition to redispersion, the product quality of vaccine suspensions can also be affected by temperature during transport. After freezing, the bond between the adjuvant and antigen may be cleaved. Separated adjuvants tend to form aggregates with larger particle size and weight, which then gradually settle to the bottom of the container. The size of the aggregates may increase after repeated freeze-thaw cycles. Aggregate formation affects both redispersion and the physical and chemical properties of the product. The WHO guidelines for international packaging and transport of vaccines include a shaker test protocol to determine whether adsorbed vaccines have been affected by freezing. The guidelines also specify that shaker tests should be performed on random samples of vaccine if there are indications that the temperature has dropped below zero during transport. On the other hand, higher temperatures may also affect the product quality of vaccine suspensions, resulting in particle formation and / or changes in chemical properties. Therefore, the WHO specifies a maximum temperature of +8°C for insulated Class A packaging during international transport for at least 48 hours. The maximum temperature allowed for Class B and C packaging is +30°C.

[0370] The following clauses describe additional aspects of the present invention: C1. A composition comprising: (a) a first polypeptide derived from a meningococcal factor H binding protein (fHBP); (b) a second polypeptide derived from a meningococcal factor H binding protein (fHBP); (c) a meningococcal serogroup A capsular saccharide conjugate; (d) a meningococcal serogroup C capsular saccharide conjugate; (e) a meningococcal serogroup W capsular saccharide conjugate; and (f) a meningococcal serogroup Y capsular saccharide conjugate, wherein the composition elicits an immune response against any one of meningococcal serogroups A, C, W-135, and Y capsular polysaccharides, and the serum bactericidal antibody response is greater than that elicited by a licensed vaccine against the meningococcal serogroups. C2. The method of clause C1, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to any one amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:62. C3. A composition comprising: (a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2; (c) a meningococcal serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (d) a meningococcal serogroup A capsular saccharide conjugated to the ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. 1. The method of clause C1, wherein the linker comprises (a) a meningococcal serogroup C capsular saccharide that is conjugated to tetanus toxoid by carbodiimide chemistry, (b) a meningococcal serogroup W capsular saccharide that is conjugated directly to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker, and (c) a meningococcal serogroup Y capsular saccharide that is conjugated directly to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker. C4. The composition elicits an immune response against any one of Neisseria meningitidis serotypes A, C, W-135, and Y, and the serum bactericidal antibody response is against any one of the approved Neisseria meningitidis serotypes A, C, W-135, and Y. The method of any one of clauses C1 to C3, wherein the IL-135 is higher than that induced by a N. meningitidis capsular polysaccharide vaccine. C5. The method of any one of clauses C1 to C3, wherein the composition elicits an immune response against meningococcal serotype A, wherein the serum bactericidal antibody response is greater than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines. C6. The method of any one of clauses C1 to C3, wherein the composition elicits an immune response against meningococcal serotype C, wherein the serum bactericidal antibody response is greater than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines. C7. The method of any one of clauses C1 to C3, wherein the composition elicits an immune response against meningococcal serotype W, wherein the serum bactericidal antibody response is greater than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines. C8. The method of any one of clauses C1 to C3, wherein the composition elicits an immune response against meningococcal serotype Y, wherein the serum bactericidal antibody response is greater than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines. C9. The method of any one of clauses C1 to C3, wherein the composition elicits an immune response against each of meningococcal serotypes A, C, W-135, and Y, wherein the serum bactericidal antibody response is greater than that elicited by licensed meningococcal serotypes A, C, W-135, and Y meningococcal capsular polysaccharide vaccines. C10. The method of any one of clauses C1 to C3, wherein the composition induces an immune response against meningococcal serogroup B, wherein the serum bactericidal antibody response is greater than that induced by a licensed meningococcal serogroup B H factor conjugate vaccine. C11. The method of any one of clauses C1 to C3, wherein the composition elicits an immune response against each of meningococcal serotypes A, C, W-135, and Y, wherein the serum bactericidal antibody response against each of meningococcal serotypes A, C, W-135, and Y capsular polysaccharide is greater than that elicited by a licensed meningococcal serotype A, C, W-135, and Y meningococcal capsular polysaccharide vaccine, and wherein the composition elicits an immune response against meningococcal serotype B, wherein the serum bactericidal antibody response is greater than that elicited by a licensed meningococcal serotype B H factor conjugate vaccine, and wherein the licensed meningococcal serotype A, C, W-135, and Y meningococcal capsular polysaccharide vaccine and the licensed meningococcal serotype B H factor conjugate vaccine are administered sequentially rather than as a combined dose. C12. The method of any one of clauses C1 to C3, wherein the composition comprises an adjuvant. C13. The method of any one of clauses C1 to C3, wherein the composition comprises an aluminum adjuvant. C14. The method of any one of clauses C1 to C3, wherein the composition comprises aluminum hydroxide. C15. The method of any one of clauses C1 to C3, wherein the composition comprises aluminum phosphate. C16. The method of any one of clauses C1 to C3, wherein the composition comprises aluminum. C17. The method of any one of clauses C1 to C3, wherein at least 90% of the first polypeptide is bound to aluminum in the composition. C18. The method of any one of clauses C1 to C3, wherein at least 90% of the second polypeptide is bound to aluminum in the composition. C19. The method of any one of clauses C1 to C3, wherein the composition is formulated as a sterile liquid. C20. The method of any one of clauses C1 to C3, wherein the composition comprises a pharmaceutically acceptable preservative. C21. The method of any one of clauses C1 to C3, wherein the composition comprises polysorbate-80. C22. The method of any one of clauses 1 to 3, wherein the composition comprises Tris-HCl, sodium chloride, sucrose, histidine, polysorbate 80, and aluminum phosphate. C23. The method of any one of clauses 1 to 3, wherein the composition comprises about 120 μg / ml of the first polypeptide, about 120 μg / ml of the second polypeptide, about 0.5 mg / ml of aluminum as aluminum phosphate, about 0.02 mg of polysorbate-80, about 10 mM histidine, and about 150 mM sodium chloride. C24. The method of any one of clauses 1 to 3, wherein the composition comprises about 60 μg of the first polypeptide, about 60 μg of the second polypeptide, about 5 μg of MenA capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenC capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenW capsular saccharide conjugated to about 3.75 μg of TT, about 5 μg of MenY capsular saccharide conjugated to about 3.25 μg of TT, about 97 μg of Tris-HCl, pH 6.8±0.3, 4.69 to 4.71 mg of sodium chloride, about 28 mg of sucrose, about 0.78 mg of L-histidine, about 0.02 mg of polysorbate-80, about 0.25 mg of aluminium, and further comprising 0.5 mL of water per dose. C25. The method of any one of clauses C1 to C3, wherein the immune response comprises serum bactericidal antibodies. C26. The method of any one of clauses C1 to C3, wherein the composition is capable of eliciting a booster immune response against at least one of meningococcal serotypes A, C, W-135, and Y. C27. The method of any one of clauses C1 to C3, wherein the composition is capable of eliciting a booster immune response against meningococcal serogroup B. C28. The method of any one of clauses C1 to C3, wherein the immune response is elicited in a human aged 10 to 26 years. C29. The method of any one of clauses C1 to C3, wherein the immune response is elicited in a human aged from 12 months to under 18 months or from 18 months to under 24 months. C30. The method of any one of clauses C1 to C3, wherein the immune response is elicited in a human aged from 18 months to under 24 months. C31. The method of any one of clauses C1 to C3, wherein the immune response is elicited in a human aged 24 months or older and less than 10 years. C32. The method of any one of clauses C1 to C3, wherein an immune response is elicited in a human who is seronegative for meningococcal serogroups A, C, W-135, and Y. C33. The method of any one of clauses C1 to C3, wherein an immune response is elicited in a human who is seropositive for meningococcal serogroups A, C, W-135, and Y. C34. The method of any one of clauses C1 to C3, wherein the composition is administered to the human in at least two doses, the second dose being about 6 months after the first dose. C35. The method of clause C34, wherein the human is at least 10 years old and at most 17 years old. C36. The method of clause C35, wherein a third dose of the composition is administered to a human, and the human is at least 16 years of age. C37. The method of any one of clauses C1 to C3, wherein the composition is administered to the human in up to two doses, the second dose being about 6 months after the first dose. C38. The method of any one of clauses C1 to C3, wherein the composition induces an immune response against A22. C39. The method of any one of clauses C1 to C3, wherein the composition induces an immune response against A56. C40. The method of any one of clauses C1 to C3, wherein the composition induces an immune response against B24. C41. Any of clauses C1 to C3, wherein the composition induces an immune response against B44. 10. The method according to claim 1. C42. The method of any one of clauses C1 to C3, wherein the composition comprises about 60 μg of the first polypeptide, about 60 μg of the second polypeptide, about 5 μg of MenA capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenC capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenW capsular saccharide conjugated to about 3.75 μg of TT, about 5 μg of MenY capsular saccharide conjugated to about 3.25 μg of TT, about 97 μg of Tris-HCl, pH 6.8±0.3, 4.69 to 4.71 mg of sodium chloride, about 28 mg of sucrose, about 0.78 mg of L-histidine, about 0.02 mg of polysorbate-80, about 0.25 mg of aluminum, and further comprising 0.5 mL of water per dose. C43. A composition comprising (a) a first polypeptide derived from a meningococcal factor H binding protein (fHBP), (b) a second polypeptide derived from a meningococcal factor H binding protein (fHBP), (c) a meningococcal serogroup A capsular glycan conjugate, (d) a meningococcal serogroup C capsular glycan conjugate, (e) a meningococcal serogroup W capsular glycan conjugate, and (f) a meningococcal serogroup Y capsular glycan conjugate, wherein the composition elicits an immune response against at least one of meningococcal serogroups A, C, W-135, and Y, and wherein the serum bactericidal antibody response is greater than that elicited by a licensed vaccine against the meningococcal serogroups. C44. The composition according to clause C43, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to any one amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:62. C45. (a) a first polypeptide derived from Neisseria meningitidis factor H binding protein (fHBP); (b) a second polypeptide derived from Neisseria meningitidis factor H binding protein (fHBP); (c) a Neisseria meningitidis serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (d) a Neisseria meningitidis serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry. 1. A composition comprising (a) a meningococcal serogroup C capsular saccharide conjugated to a tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker; (b) a meningococcal serogroup W capsular saccharide conjugated to a tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker; and (c) a meningococcal serogroup Y capsular saccharide conjugated to a tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker; wherein the composition elicits an immune response against each of the meningococcal serogroups A, C, W-135 and Y capsular polysaccharides, and the serum bactericidal antibody response is greater than that elicited by a licensed meningococcal serogroup A, C, W-135 and Y meningococcal capsular polysaccharide vaccine. C46. A method of inducing an immune response in a human against meningococcal serogroup B subfamily A strains and meningococcal serogroup B subfamily B strains, comprising administering to the human an effective amount of the composition of any one of clauses C43 to C45. C47. A method of inducing an immune response in a human against meningococcal serogroup A, meningococcal serogroup C, meningococcal serogroup W, and / or meningococcal serogroup Y strains, comprising administering to the human an effective amount of the composition of any one of clauses C43 to C45. C48. A method of inducing an immune response in a human against meningococcal serogroup A, meningococcal serogroup B, meningococcal serogroup C, meningococcal serogroup W, and / or meningococcal serogroup Y strains, comprising administering to the human an effective amount of the composition of any one of clauses C43 to C45. C49. A method for preventing meningococcal serogroup A, meningococcal serogroup B, meningococcal serogroup C, meningococcal serogroup D, meningococcal serogroup E, meningococcal serogroup F, meningococcal serogroup G, meningococcal serogroup H, meningococcal serogroup I ... and a method for inducing an immune response against Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W, Neisseria meningitidis serogroup Y strains, and / or Neisseria meningitidis serogroup X strains. C50. The method of any one of clauses C46 to C49, wherein the patient has not previously received a multivalent meningococcal capsular saccharide-carrier protein conjugate vaccine prior to the first administration of the composition of any one of clauses C43 to C45. C51. The method of any one of clauses C46 to C49, wherein the patient has previously received a multivalent meningococcal capsular saccharide-carrier protein conjugate vaccine prior to the first administration of the composition of any one of clauses C1 and C10. C52. Use of an effective amount of a composition for inducing an immune response to meningococcal serogroup B in a human, wherein the composition comprises: a) a first lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; and b) a second lipidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, and wherein the composition induces an immune response to at least one meningococcal serogroup B strain expressing a polypeptide selected from the group consisting of A02, A28, A42, A63, A76, B05, B07, B08, B13, B52, and B107. C53. The use according to clause C52, wherein the induced immune response is bactericidal. C54. The use according to clause C52, wherein the composition further comprises polysorbate-80. C55. The use of any one of clauses C52 to C54, wherein the composition further comprises aluminum. C56. The use of any one of clauses C52 to C55, wherein the composition further comprises a histidine buffer. C57. The use of any one of clauses C52 to C56, wherein the composition further comprises sodium chloride. C58. The use of any one of clauses C52 to C57, wherein the composition comprises about 120 μg / ml of the first polypeptide, about 120 μg / ml of the second polypeptide, polysorbate-80 at a molar ratio of about 2.8, about 0.5 mg / ml of aluminum, about 10 mM histidine, and about 150 mM sodium chloride. C59. The use of any one of clauses C52 to C58, wherein the composition comprises about 60 μg of the first polypeptide, about 60 μg of the second polypeptide, about 18 μg of polysorbate-80, about 250 μg of aluminum, about 780 μg of histidine, and about 4380 μg of sodium chloride. C60. The composition further comprises at least one additional immunogenic composition comprising a mixture of four distinct and separately produced protein-capsular polysaccharide conjugates, wherein a first conjugate comprises a meningococcal capsular polysaccharide of serotype W conjugated to a carrier protein, a second conjugate comprises a meningococcal capsular polysaccharide of serotype Y conjugated to a carrier protein, a third conjugate comprises a meningococcal capsular polysaccharide of serotype A conjugated to a carrier protein, and a fourth conjugate comprises a meningococcal capsular polysaccharide of serotype C conjugated to a carrier protein, wherein the carrier protein is selected from the group consisting of diphtheria toxoid, CRM 197 The use according to any one of clauses C52 to C59, wherein the antibody is selected from the group consisting of: C61. The use according to clause C60, wherein the carrier protein is diphtheria toxoid. C62. The use according to clause C60, wherein the carrier protein is tetanus toxoid. C63. The use according to clause C60, wherein the at least one additional immunogenic composition is a liquid composition. C64. The use according to clause C60, wherein the at least one additional immunogenic composition is not lyophilized. C65. The use of any one of clauses C60 to C64, wherein the composition induces an immune response against at least one meningococcal serogroup A strain. C66. The use of any one of clauses C60 to C64, wherein the composition induces an immune response against at least one meningococcal serogroup C strain. C67. The composition induces an immune response against at least one meningococcal serogroup W strain. 10. The use according to any one of clauses C60 to C64. C68. The use of any one of clauses C60 to C64, wherein the composition induces an immune response against at least one meningococcal serotype Y strain. C69. The use of any one of clauses C60 to C64, wherein the composition induces an immune response against at least one of Neisseria meningitidis serogroup A strains, Neisseria meningitidis serogroup C strains, Neisseria meningitidis serogroup Y strains, Neisseria meningitidis serogroup W strains, and any combination thereof. C70. The use of any one of clauses C52 to C69, wherein the effective amount of the composition comprises a single dose. C71. The use of any one of clauses C52 to C70, wherein the effective amount of the composition comprises two doses. C72. The use of any one of clauses C52 to C70, wherein the effective amount of the composition further comprises a booster dose. C73. The use of any one of clauses C52 to C70, wherein the effective amount of the composition comprises a maximum of two doses. C74. The use of any one of clauses C52 to C70, wherein the effective amount of the composition comprises up to three doses. C75. The use of clause C52, wherein the composition does not comprise a hybrid protein. C76. The use of clause C52, wherein the composition does not comprise a fusion protein. C77. The use according to clause C52, wherein the composition is not lyophilized. C78. Use according to clause C52, wherein the composition is formaldehyde-free. C79. The use according to clause C60, wherein the composition does not comprise diphtheria toxoid or CRM. C80. Neisseria meningitidis serogroup A (MenA) capsular saccharide is conjugated to an adipic acid dihydrazide (ADH) linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, and the linker is conjugated to the tetanus toxoid carrier protein (TT) via carbodiimide chemistry (MenA). AH -TT conjugate), for use as described in clause C60. C81. Meningococcal serogroup C (MenC) capsular saccharide conjugated to an ADH linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, and the linker conjugated to the tetanus toxoid carrier protein (TT) via carbodiimide chemistry (MenC AH -TT conjugate), for use as described in clause C60. C82. The use according to clause C60, wherein the meningococcal serogroup W (MenW) capsular saccharide is directly conjugated to the tetanus toxoid carrier protein (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate). C83. The use according to clause C60, wherein the meningococcal serogroup Y (MenY) capsular saccharide is directly conjugated to the tetanus toxoid carrier protein (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate). C84. Meningococcal serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, and the linker conjugated to tetanus toxoid carrier protein (TT) via carbodiimide chemistry (MenA AH -TT conjugate), in which Neisseria meningitidis serogroup C (MenC) capsular saccharide is conjugated to an ADH linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, and the linker is conjugated to the tetanus toxoid carrier protein (TT) via carbodiimide chemistry (MenC AHIn the absence of a linker, meningococcal serotype W (MenW) capsular saccharides are directly conjugated to the tetanus toxoid carrier protein (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry (MenW-TT conjugate), and in the absence of a linker, meningococcal serotype Y (MenY ) The use according to clause C60, wherein the capsular saccharide is directly conjugated to the tetanus toxoid carrier protein (TT) by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry (MenY-TT conjugate). C85. The use of clause C60, wherein the composition does not contain MenA capsular saccharides in the absence of an adipic acid dihydrazide (ADH) linker. C86. The use of any one of clauses C52 to C85, wherein the patient is aged between 12 and under 18 months or between 18 and under 24 months. C87. The use of any one of clauses C52 to C85, wherein the patient is aged from 18 months to less than 24 months. C88. The use of any one of clauses C52 to C85, wherein the patient is at least 24 months old and less than 10 years old. C89. The use of any one of clauses C52 to C88, wherein the composition induces a serum immunoglobulin bactericidal titer in a human after receiving the first dose that is at least two-fold higher than the serum immunoglobulin bactericidal titer in the human before receiving the first dose, when measured in a serum bactericidal assay using human complement under the same conditions. C90. The use of any one of clauses C52 to C89, wherein the composition induces a serum immunoglobulin bactericidal titer in a human after receiving the first dose that is at least four times higher than the serum immunoglobulin bactericidal titer in the human before receiving the first dose, when measured in a serum bactericidal assay using human complement under the same conditions. C91. The use of any one of clauses C52 to C90, wherein the composition induces a serum immunoglobulin bactericidal titer in a human after receiving the first dose that is at least 8-fold higher than the serum immunoglobulin bactericidal titer in the human before receiving the first dose, when measured in a serum bactericidal assay using human complement under the same conditions. C92. A method of inducing an immune response in a human, the method comprising administering to the human: a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2; (c) a meningococcal serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (d) a meningococcal serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry. (e) a meningococcal serogroup C capsular saccharide conjugated directly to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker; and (f) a meningococcal serogroup Y capsular saccharide conjugated directly to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker, wherein the composition induces an immune response against at least one of meningococcal serogroups A, C, W-135 and Y, the immune response comprising titers of serum bactericidal antibodies which are higher than those induced by licensed vaccines against meningococcal serogroups A, C, W-135 and Y meningococcal capsular polysaccharides. C93. The method of claim 1, wherein the licensed vaccine against meningococcal serogroups A, C, W-135, and Y meningococcal capsular polysaccharide is MENVEO. C94. A method of inducing an immune response in a human, the method comprising administering to the human: a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2; (c) a meningococcal serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (d) a meningococcal serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is carbodiimide. (e) a meningococcal serogroup W capsular saccharide that is directly conjugated to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker; and (f) a meningococcal serogroup Y capsular saccharide that is directly conjugated to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker, wherein the composition induces an immune response against meningococcal serogroup B, the immune response comprising a titer of serum bactericidal antibodies that is higher than the titer of serum bactericidal antibodies induced by a licensed vaccine against meningococcal serogroup B. C95. The method of clause C94, wherein the licensed vaccine against meningococcal serogroup B is TRUMENBA. C96. A method of inducing an immune response in a human, the method comprising administering to the human: a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2; (c) a meningococcal serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; (d) a meningococcal serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (e) a meningococcal serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, in the absence of a linker. and (f) a meningococcal serogroup Y capsular saccharide that is directly conjugated to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker, wherein the composition is selected from the group consisting of meningococcal serogroups A, C, W-135, and Y and meningococcal serogroup B. and inducing an immune response against one of the N. meningitidis serotypes A, C, W-135, and Y capsular polysaccharides, wherein the immune response comprises a serum bactericidal antibody titer against each of the N. meningitidis serotypes A, C, W-135, and Y that is higher than the serum bactericidal antibody titer induced by a licensed vaccine against N. meningitidis serotype B, and wherein the immune response comprises a serum bactericidal antibody titer against N. meningitidis serotype B that is higher than the serum bactericidal antibody titer induced by a licensed vaccine against N. meningitidis serotype B. C97. The method of clause C96, wherein the licensed vaccine against meningococcal serogroups A, C, W-135, and Y meningococcal capsular polysaccharide is MENVEO. C98. The method of any one of clauses C94 to C97, wherein the composition further comprises an adjuvant. C99. The method of any one of clauses C94 to C97, wherein the composition further comprises aluminum. C100. The method of clause C99, wherein the adjuvant comprises aluminum hydroxide. C101. The method of clause C99, wherein the adjuvant comprises aluminum phosphate. C102. The method of clause C94, wherein at least 90% of the first polypeptide is bound to aluminum in the composition. C103. The method of clause C94, wherein at least 90% of the second polypeptide is bound to aluminum in the composition. C104. The method of any one of clauses C94 to C97, wherein the composition is formulated as a sterile liquid. C105. The method of any one of clauses C94 to C97, wherein the composition further comprises polysorbate-80. C106. The method of any one of clauses C94 to C97, wherein the composition further comprises Tris-HCl, sodium chloride, sucrose, histidine, polysorbate 80, and aluminum phosphate. C107. The composition comprises about 120 μg / ml of a first polypeptide, about 120 μg / ml of a The method of any one of clauses C94 to C97, comprising a second polypeptide, about 0.5 mg / ml aluminum as aluminum phosphate, about 0.02 mg polysorbate-80, about 10 mM histidine, and about 150 mM sodium chloride. C108. The method of any one of clauses C94 to C97, wherein the composition comprises about 60 μg of the first polypeptide, about 60 μg of the second polypeptide, about 5 μg of MenA capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenC capsular saccharide conjugated to about 7.5 μg of TT, about 5 μg of MenW capsular saccharide conjugated to about 3.75 μg of TT, about 5 μg of MenY capsular saccharide conjugated to about 3.25 μg of TT, about 97 μg of Tris-HCl, pH 6.8±0.3, 4.69 to 4.71 mg of sodium chloride, about 28 mg of sucrose, about 0.78 mg of L-histidine, about 0.02 mg of polysorbate-80, about 0.25 mg of aluminum, and further comprising 0.5 mL of water per dose. C109. The method of any one of clauses C94 to C97, wherein the composition is capable of eliciting a booster immune response against each of meningococcal serotypes A, C, W-135, and Y. C110. The method of any one of clauses C94 to C97, wherein the composition is capable of eliciting a booster immune response against meningococcal serogroup B. C111. The method of any one of clauses C94 to C97, wherein the human is aged 10 to 26 years. C112. The method of any one of clauses C94 to C97, wherein the human is aged between 12 and under 18 months or between 18 and under 24 months. C113. The method of any one of clauses C94 to C97, wherein the human is aged between 18 months and less than 24 months. C114. The method of any one of clauses C94 to C97, wherein the human is at least 24 months old and less than 10 years old. C115. The method of any one of clauses C94 to C97, wherein the human is at least 16 years old. C116. The method of clause C115, comprising administering a single dose to a human. C117. The method of any one of clauses C94 to C97, wherein the human is 10 to 12 years old. C118. The method of clause C113, comprising administering at least two doses to a human. C119. The method of clause C113, wherein the human is at most 16 years old. C120. The method of any one of clauses C94 to C97, comprising administering at least one dose of the composition to a human at about 11 years of age, and administering an additional dose of the composition to the human at least 4 years after the first dose. C121. The method of any one of clauses C94 to C97, comprising administering at least one dose of the composition to a human at about 11 years of age, and administering an additional dose of the composition to the human at least 4 years after the last dose. C122. The method of clause C116, wherein an additional dose of the composition is administered about 5 years after the last dose. C123. The method of any one of clauses C94 to C97, comprising administering one dose of the composition to a human at about 11 years of age, and about 5 years after the first dose, administering at least two doses of the composition to the human. C124. The method of any one of clauses C94 to C97, wherein the human is seronegative for meningococcal serogroups A, C, W-135, and Y capsular polysaccharides. C125. The method of any one of clauses C94 to C97, wherein the human is seropositive for meningococcal serogroups A, C, W-135, and Y capsular polysaccharides. Administering a first dose and a second dose of the C126 composition, wherein the second dose is about 6 months after the first dose, according to any one of clauses C94 to C97. method. C127. The method of clause C122, wherein the human is at most 17 years old. C128. The method of clause C122, comprising administering a third dose of the composition to a human being who is 16 years of age. C129. The method of any one of clauses C94 to C97, comprising administering up to two doses of the composition, the second dose being about 6 months after the first dose. C130. The method of any one of clauses C94 to C97, wherein the composition elicits an immune response against a meningococcal serogroup B strain that expresses A22. C131. The method of any one of clauses C94 to C97, wherein the composition elicits an immune response against a meningococcal serogroup B strain that expresses A56. C132. The method of any one of clauses C94 to C97, wherein the composition elicits an immune response against a meningococcal serogroup B strain expressing B24. C133. The method of any one of clauses C94 to C97, wherein the composition elicits an immune response against a meningococcal serogroup B strain expressing B44. C134. The method of any one of clauses C94 to C97, wherein the composition induces a bactericidal immune response against any one of Neisseria meningitidis serogroup B A22, A56, B24, B44 strains, or any combination thereof. C135. The method of any one of clauses C94 to C97, wherein the composition induces a bactericidal immune response against any one of Neisseria meningitidis serogroup B B24, B16, B44, A22, B03, B09, A12, A19, A05, A07, B153 strains, or any combination thereof.

Claims

1. 1. A method of inducing an immune response in a human, the method comprising administering to the human: (a) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2; (c) a meningococcal serogroup A capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; (d) a meningococcal serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to tetanus toxoid by carbodiimide chemistry; and (e) a meningococcal serogroup C capsular saccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, in the absence of a linker. (f) a composition comprising a meningococcal serogroup W capsular saccharide directly conjugated to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, in the absence of a linker, and a meningococcal serogroup Y capsular saccharide directly conjugated to tetanus toxoid by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, in the absence of a linker, wherein the composition induces an immune response against at least one of meningococcal serogroups A, C, W-135 and Y; The immune response comprises a titer of serum bactericidal antibodies, the titer being greater than that induced by a composition selected from any one of: (a) a composition comprising a meningococcal serogroup A, C, W-135, and Y meningococcal capsular polysaccharide conjugate and not further comprising a meningococcal polypeptide; and (b) a composition comprising a meningococcal serogroup B polypeptide and not further comprising a meningococcal serogroup A, C, W-135, and Y meningococcal capsular polysaccharide conjugate.

2. 10. The method of claim 1, wherein the composition comprising a meningococcal serogroup A, C, W-135, and Y meningococcal capsular polysaccharide conjugate is a licensed vaccine against meningococcal serogroup A, C, W-135, and Y meningococcal capsular polysaccharide.

3. 3. The method of any one of claims 1 to 2, wherein the licensed vaccine against meningococcal serogroups A, C, W-135, and Y meningococcal capsular polysaccharides is MENVEO.

4. 4. The method of claim 1, wherein the composition comprising a meningococcal serogroup B polypeptide is a licensed vaccine against meningococcal serogroup B.

5. 5. The method of any one of claims 1 to 4, wherein the licensed vaccine against meningococcal serogroup B is TRUMENBA.

6. 6. The method of any one of claims 1 to 5, wherein the composition induces an immune response against a meningococcal serotype selected from any one of meningococcal serotypes A, C, W-135, and Y and meningococcal serotype B, wherein the immune response comprises a serum bactericidal antibody titer against the selected meningococcal serotype that is higher than the serum bactericidal antibody titer induced by a composition against meningococcal serotypes A, C, W-135, and Y capsular polysaccharides.

7. 7. The method of any one of claims 1 to 6, wherein the composition induces an immune response against a meningococcal serotype selected from any one of meningococcal serotypes A, C, W-135, and Y and meningococcal serotype B, wherein the immune response comprises a serum bactericidal antibody titer against the selected meningococcal serotype that is higher than the serum bactericidal antibody titer induced by the composition against meningococcal serotype B.

8. The method of claim 1 , wherein the composition further comprises an adjuvant. 。

9. 9. The method of claim 1, wherein the composition further comprises aluminum.

10. 10. The method of claim 1, wherein the adjuvant comprises aluminum hydroxide.

11. 11. The method of claim 1, wherein the adjuvant comprises aluminum phosphate.

12. 12. The method of claim 1, wherein at least 90% of the first polypeptide is bound to aluminum in the composition.

13. 13. The method of any one of claims 1 to 12, wherein at least 90% of the second polypeptide is bound to aluminum in the composition.

14. 14. The method of any one of claims 1 to 13, wherein the composition is formulated as a sterile liquid.

15. 15. The method of any one of claims 1 to 14, wherein the composition further comprises polysorbate-80.

16. 16. The method of any one of claims 1 to 15, wherein the composition further comprises Tris-HCl, sodium chloride, sucrose, histidine, polysorbate 80, and aluminum phosphate.

17. 17. The method of any one of claims 1 to 16, wherein the composition comprises about 120 μg / ml of the first polypeptide, about 120 μg / ml of the second polypeptide, about 0.5 mg / ml aluminum as aluminum phosphate, about 0.02 mg polysorbate-80, about 10 mM histidine, and about 150 mM sodium chloride.

18. the composition comprising about 60μg of said first polypeptide, about 60μg of said second polypeptide conjugated to about 7.5μg TT, about 5μg MenA capsular saccharide conjugated to about 7.5μg TT, about 5μg MenC capsular saccharide conjugated to about 7.5μg TT, about 5μg MenW capsular saccharide conjugated to about 3.75μg TT, about 3.25μg TT 18. The method of any one of claims 1 to 17, comprising about 5µg MenY capsular saccharide conjugated to T, about 97µg Tris-HCl, pH 6.8±0.3, 4.69-4.71 mg sodium chloride, about 28 mg sucrose, about 0.78 mg L-histidine, about 0.02 mg polysorbate-80, about 0.25 mg aluminium, and further comprising 0.5 mL water per dose.

19. 19. The method of any one of claims 1 to 18, wherein the composition is capable of eliciting a booster immune response against each of meningococcal serotypes A, C, W-135, and Y.

20. 20. The method of any one of claims 1 to 19, wherein the composition is capable of eliciting a booster immune response against meningococcal serogroup B.

21. 21. The method of any one of claims 1 to 20, wherein the human is aged between 10 and 26 years.

22. 22. The method of any one of claims 1 to 21, wherein the human is 10 to 12 years old.

23. 23. The method of any one of claims 1 to 22, comprising administering at least two doses to the human.

24. 24. The method of any one of claims 1 to 23, wherein the human is at most 16 years old.

25. 25. The method of any one of claims 1 to 24, comprising administering at least one dose of the composition to the human at about 11 years of age, and administering an additional dose of the composition to the human at least four years after the first dose.

26. 26. The method of any one of claims 1 to 25, comprising administering at least one dose of the composition to the human at about 11 years of age, and administering an additional dose of the composition to the human at least 4 years after the last dose.

27. 27. The method of any one of claims 1 to 26, wherein the further dose of the composition is administered about 5 years after the last dose.

28. 28. The method of any one of claims 1-27, comprising administering one dose of the composition to the human at about 11 years of age, and about 5 years after the first dose, administering at least two doses of the composition to the human.

29. 29. The method of any one of claims 1 to 28, wherein the human is seronegative for meningococcal serogroups A, C, W-135, and Y capsular polysaccharides.

30. 30. The method of any one of claims 1 to 29, wherein the human is seropositive for meningococcal serogroups A, C, W-135, and Y capsular polysaccharides.

31. 31. The method of any one of claims 1 to 30, comprising administering a first dose and a second dose of the composition, wherein the second dose is about 6 months after the first dose.

32. 32. The method of any one of claims 1 to 31, comprising administering to the human a third dose of the composition by age 16.

33. 33. The method of any one of claims 1-32, comprising administering up to two doses of the composition, the second dose being about six months after the first dose.

34. 34. The method of any one of claims 1 to 33, wherein the composition elicits an immune response against meningococcal serogroup B strains that express A22.

35. 35. The method of any one of claims 1 to 34, wherein the composition elicits an immune response against meningococcal serogroup B strains expressing A56.

36. 36. The method of any one of claims 1 to 35, wherein the composition elicits an immune response against meningococcal serogroup B strains that express B24.

37. 37. The method of any one of claims 1 to 36, wherein the composition elicits an immune response against meningococcal serogroup B strains that express B44.

38. 38. The method of any one of claims 1 to 37, wherein the composition induces a bactericidal immune response against any one of Neisseria meningitidis serogroup B strains A22, A56, B24, B44, or any combination thereof.

39. 39. The method of any one of claims 1 to 38, wherein the composition induces a bactericidal immune response against any one of meningococcal serogroup B B24, B16, B44, A22, B03, B09, A12, A19, A05, A07, B153 strains, or any combination thereof.

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