Neisseria meningitidis vaccine

Abstract

The present application relates to Neisseria meningitidis vaccines, and specifically provides compounds, compositions, formulations, kits, uses and methods for vaccinating a subject against Neisseria meningitidis.

Description

[0001] This invention application is a divisional application based on an invention patent application with application date of September 1, 2017, application number 201780066432.4 (international application number PCT / US2017 / 049856) and name “Neisseria meningitidis vaccine”. Technical Field

[0002] The present application relates to Neisseria meningitidis vaccines, and specifically provides compounds, compositions, formulations, kits, uses and methods for vaccinating a subject against Neisseria meningitidis. Background Art

[0003] Neisseria meningitidis (N. meningitidis) is a major cause of bacterial meningitis and sepsis worldwide. Serogroups A, C, Y, and W-135 (MenA, MenC, MenY, and MenW, respectively; collectively MenACYW) of N. meningitidis are responsible for a large proportion of meningococcal disease worldwide. Six types of vaccines are currently available to protect against N. meningitidis—quadrivalent meningococcal conjugate vaccines, such as and Meningococcal polysaccharide vaccines, such as Serogroup C meningococcal vaccine, such as and Serogroup A meningococcal vaccine, such as Serogroup C and Y meningococcal vaccines, such as and serogroup B meningococcal vaccines, such as and

[0004] The epidemiology of Neisseria meningitidis can be described as complex, unpredictable, geographically diverse, and changing over time. Therefore, there is a need to develop improved Neisseria meningitidis vaccines. Specifically, existing polysaccharide conjugate vaccines may not be suitable for administration to one or more of infants, young children, adolescents, and / or the elderly, or may result in weak or undetectable serological responses in some recipients. Summary of the invention

[0005] In some embodiments, the compositions, methods and / or uses disclosed herein provide one or more benefits, or at least provide the public with a useful choice. These benefits may include one or more of the following: improved immunogenicity for one, two, three or all four of MenA, MenC, MenY and MenW; improved immunogenicity in two, three or four categories in infants, young children, adolescents and the elderly; and sufficient stability to allow long-term storage as a liquid formulation, for example, for many years (e.g., 2.5, 3, 3.5, 4 or 4.5 years) under refrigeration or for many months (e.g., 2, 3, 4, 5 or 6 months) at room temperature.

[0006] Therefore, the following embodiments are provided. Embodiment 1 is a Neisseria meningitidis vaccine composition comprising: a) a first conjugate of MenA capsular polysaccharide and a carrier protein; b) a second conjugate of MenC capsular polysaccharide and a carrier protein; c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein the second conjugate is a population comprising a double-terminal-linked conjugated polysaccharide and a single-terminal-linked conjugated polysaccharide both attached to the carrier protein via a secondary amine, and the O-acetylation level of the polysaccharide of the second conjugate is 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide. The O-acetylation level may be greater than or equal to 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2 μmol / mg polysaccharide. The O-acetylation level may be less than or equal to 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 μmol / mg polysaccharide. For example, the level may range from 0.6 to 1.5 μmol / mg polysaccharide or from 0.8 to 1.4 μmol / mg polysaccharide. The O-acetyl content may be measured by the Hestrin method (Hestrin et al., J. Biol. Chem. 1949, 180, p. 249).

[0007] Embodiment 2 is a Neisseria meningitidis vaccine composition comprising: a) a first conjugate of MenA capsular polysaccharide and a carrier protein; b) a second conjugate of MenC capsular polysaccharide and a carrier protein; c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein the second conjugate is a population comprising single-end-linked conjugated polysaccharides attached to the carrier protein via a secondary amine, wherein the single-end-linked conjugated polysaccharide has a terminal unlinked sugar, wherein the terminal sugar has a primary hydroxyl or secondary amine linkage at position 7, or wherein the reducing end is modified by a (2-hydroxy)ethoxy or secondary amine linkage. The second conjugate population may also comprise double-end-linked conjugates in which the carrier protein is linked to both ends of the polysaccharide (e.g., via a secondary amine).

[0008] Embodiment 3 is a Neisseria meningitidis vaccine composition comprising: a) a first conjugate of MenA capsular polysaccharide and a carrier protein; b) a second conjugate of MenC capsular polysaccharide and a carrier protein; c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein the MenA capsular polysaccharide is attached to the carrier protein via a linker comprising a carbamate, a spacer and an amide, wherein the spacer is between the carbamate and the amide and comprises 2-10 linear carbons, and the polysaccharide to carrier protein mass ratio of the first conjugate is 0.3 to 1.5. The polysaccharide to carrier protein mass ratio of the first conjugate can be, for example, 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, or 1.4 to 1.5.

[0009] Embodiment 4 is a Neisseria meningitidis vaccine composition comprising: a) a first conjugate of MenA capsular polysaccharide and a carrier protein; b) a second conjugate of MenC capsular polysaccharide and a carrier protein; c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein the MenA capsular polysaccharide is attached to the carrier protein via a linker comprising a carbamate, a spacer and an amide, wherein the spacer is between the carbamate and the amide and comprises 2-10 linear carbons; and wherein the MenC, MenW-135 and MenY capsular polysaccharides are attached to the carrier protein via secondary amines; and the weight average molecular weight of at least one of the conjugates ranges from 300 kDa to 1500 kDa.

[0010] Embodiment 5 is a Neisseria meningitidis vaccine composition comprising: a) a first conjugate of MenA capsular polysaccharide and a carrier protein; b) a second conjugate of MenC capsular polysaccharide and a carrier protein; c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein the carrier protein is tetanus toxoid; one or more of the first, second, third and fourth conjugates have a weight average molecular weight in the range of 300 kDa to 1500 kDa; and the composition contains less than 20% by weight of free polysaccharide relative to the total polysaccharide. In the composition, at least one, two, three or four of the conjugates may have a weight average molecular weight in the range of 300 kDa to 1500 kDa. In the composition, the weight average molecular weight of at least one, two, three or four of the conjugates may be greater than or equal to 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, or 1100 kDa. In the composition, the weight average molecular weight of at least one, two, three or four of the conjugates may be less than or equal to 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa or 1400 kDa. The molecular weight of at least one conjugate may be 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400 or 1400-1500 kDa. The molecular weight of the MenA conjugate may be 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400 or 1400-1500 kDa. The molecular weight of the MenC conjugate may be 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400 or 1400-1500 kDa. The molecular weight of the MenY conjugate may be 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400 or 1400-1500 kDa. The molecular weight of the MenW-135 conjugate may be 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400 or 1400-1500 kDa.

[0011] Embodiment 6 is a Neisseria meningitidis vaccine composition comprising: a) a first conjugate of MenA capsular polysaccharide and a carrier protein; b) a second conjugate of MenC capsular polysaccharide and a carrier protein; c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein the carrier protein is tetanus toxoid; the polysaccharide to carrier protein mass ratio of one or more of the first, second, third and fourth conjugates is from 0.3 to 1.5; and the composition comprises less than 20% by weight of free polysaccharide relative to the total polysaccharide. The polysaccharide to carrier protein mass ratio of the first conjugate can be, for example, 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, or 1.4 to 1.5. The polysaccharide to carrier protein mass ratio of the second conjugate can be, for example, 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, or 1.4 to 1.5. The polysaccharide to carrier protein mass ratio of the third conjugate can be, for example, 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, or 1.4 to 1.5. The polysaccharide to carrier protein mass ratio of the fourth conjugate can be, for example, 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, or 1.4 to 1.5.

[0012] Embodiment 7 is the vaccine composition of any one of the preceding embodiments, wherein the first, second, third and / or fourth conjugate is a population comprising molecules with a molecular weight in the range of 700kDa to 1400kDa or 800kDa to 1300kDa. The first and second conjugates may be populations comprising molecules with a molecular weight in the range of 700kDa to 1400kDa or 800kDa to 1300kDa. The first and third conjugates may be populations comprising molecules with a molecular weight in the range of 700kDa to 1400kDa or 800kDa to 1300kDa. The first and fourth conjugates may be populations comprising molecules with a molecular weight in the range of 700kDa to 1400kDa or 800kDa to 1300kDa. The second and third conjugates may be populations comprising molecules with a molecular weight in the range of 700kDa to 1400kDa or 800kDa to 1300kDa. The second and fourth conjugates may be populations comprising molecules with molecular weights ranging from 700 kDa to 1400 kDa or from 800 kDa to 1300 kDa.The third and fourth conjugates may be populations comprising molecules with molecular weights ranging from 700 kDa to 1400 kDa or from 800 kDa to 1300 kDa.

[0013] Embodiment 8 is a Neisseria meningitidis vaccine composition comprising a conjugate of a MenC capsular polysaccharide and a carrier protein, wherein the conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide both attached to the carrier protein via a secondary amine, and the O-acetylation level of the polysaccharide of the conjugate of the MenC capsular polysaccharide and the carrier protein ranges from 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide. The O-acetylation level may be greater than or equal to 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2 μmol / mg polysaccharide. The O-acetylation level may be less than or equal to 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 μmol / mg polysaccharide. For example, the level may be 0.6 to 1.5 μmol / mg polysaccharide or 0.8 to 1.4 μmol / mg polysaccharide. The O-acetyl content can be measured by the Hestrin method (Hestrin et al., J. Biol. Chem. 1949, 180, p. 249).

[0014] Embodiment 9 is the vaccine composition of embodiment 8, wherein the conjugate (a) has a weight average molecular weight ranging from 300 kDa to 1500 kDa; or (b) is a population comprising molecules with a molecular weight ranging from 700 kDa to 1400 kDa or 800 kDa to 1300 kDa.

[0015] Embodiment 10 is the vaccine composition of any one of embodiments 4, 5, 7 or 9, wherein the molecular weight is determined by multi-angle light scattering (MALS).

[0016] Embodiment 11 is the vaccine composition of any one of the preceding embodiments, wherein the degree of O-acetylation of the MenC polysaccharide ranges from 0.6 to 1.5 μmol / mg polysaccharide or from 0.8 to 1.4 μmol / mg polysaccharide.

[0017] Embodiment 12 is the vaccine composition of embodiment 11, wherein the degree of O-acetylation is greater than or equal to 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2 μmol / mg polysaccharide.

[0018] Embodiment 13 is the vaccine composition of embodiment 11, wherein the degree of O-acetylation is less than or equal to 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 or 1.4 μmol / mg polysaccharide.

[0019] Embodiment 14 is the vaccine composition of any one of the preceding embodiments, wherein the conjugate comprising MenC polysaccharide is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide.

[0020] Embodiment 15 is the vaccine composition of embodiment 14, wherein the single-end-linked polysaccharide of the second conjugate comprises a terminal unlinked sugar, wherein the single-end-linked conjugated polysaccharide has a terminal unlinked sugar, wherein the terminal sugar has a primary hydroxyl group at position 7, or wherein the reducing end is modified with a (2-hydroxy)ethoxy group.

[0021] Embodiment 16 is the vaccine composition of any one of the preceding embodiments, wherein the conjugate comprising MenC polysaccharide comprises one or more modifications selected from the group consisting of: (i) a primary hydroxyl group at position 7, (ii) a (2-hydroxy)ethoxy group at the reducing end, and (iii) conjugation to the carrier protein, wherein the modification is present at no less than 25 nmol / mg polysaccharide.

[0022] Embodiment 17 is the vaccine composition of any one of the preceding embodiments, comprising a conjugate of MenW-135 and / or MenY polysaccharide comprising one or more modifications selected from: (i) primary hydroxyl groups at vicinal diol positions in native MenW-135 or MenY polysaccharide and (ii) conjugation to the carrier protein, wherein the modifications are present at not less than 60 nmol / mg polysaccharide.

[0023] Embodiment 18 is the vaccine composition of embodiment 16 or 17, wherein the modification is present in an amount of less than 200 nmol / mg polysaccharide, less than 150 nmol / mg polysaccharide, less than 150 nmol / mg polysaccharide, less than 100 nmol / mg polysaccharide or less than 80 nmol / mg polysaccharide.

[0024] Embodiment 19 is the vaccine composition of any one of the preceding embodiments, wherein the size of the MenC polysaccharide is reduced by 3-fold to 8-fold relative to the native MenC polysaccharide.

[0025] Embodiment 20 is the vaccine composition of any one of the preceding embodiments, comprising a conjugate of MenA capsular polysaccharide and a carrier protein, the polysaccharide to carrier protein mass ratio of the conjugate being 0.5 to 1.5. Embodiment 20A is the vaccine composition of embodiment 20, comprising a conjugate of MenA capsular polysaccharide and a carrier protein, the polysaccharide to carrier protein mass ratio of the conjugate being 0.7 to 1.4.

[0026] Embodiment 21 is the vaccine composition of embodiment 20, wherein the polysaccharide to carrier protein mass ratio of the MenA conjugate is 0.8 to 1.3.

[0027] Embodiment 22 is the vaccine composition of any one of the preceding embodiments, comprising a conjugate of MenC and / or MenY capsular polysaccharide and a carrier protein, the polysaccharide to carrier protein mass ratio of the conjugate being 0.3 to 1.1.

[0028] Embodiment 23 is the vaccine composition of embodiment 22, wherein the polysaccharide to carrier protein mass ratio of the MenC conjugate is 0.4 to 0.8.

[0029] Embodiment 24 is the vaccine composition of any one of the preceding embodiments, comprising a conjugate of MenW-135 capsular polysaccharide and a carrier protein, wherein the polysaccharide to carrier protein mass ratio of the conjugate is 0.3 to 1.3.

[0030] Embodiment 25 is the vaccine composition of embodiment 24, wherein the polysaccharide to carrier protein mass ratio of the MenW-135 conjugate is 0.6 to 1.3.

[0031] Embodiment 26 is the vaccine composition of any one of the preceding embodiments, comprising a conjugate of MenY capsular polysaccharide and a carrier protein, the polysaccharide to carrier protein mass ratio of the conjugate being 0.5 to 1.3.

[0032] Embodiment 27 is the vaccine composition of embodiment 26, wherein the polysaccharide to carrier protein mass ratio of the MenY conjugate is 0.5 to 0.9.

[0033] Embodiment 28 is the vaccine composition of any one of the preceding embodiments, wherein the composition comprises less than 20% by weight of free polysaccharides.

[0034] Embodiment 29 is the vaccine composition of embodiment 28, wherein the composition comprises less than 10% by weight free polysaccharide, less than 5% by weight free polysaccharide, or is substantially free of free polysaccharide.

[0035] Embodiment 30 is the vaccine composition of any one of the preceding embodiments, wherein the polysaccharide of the MenA, MenC, MenW-135 or MenY conjugate is attached to the carrier protein via a linker.

[0036] Embodiment 31 is the vaccine composition of embodiment 30, wherein the linker comprises 2-10 linear carbons.

[0037] Embodiment 32 is the vaccine composition of embodiments 30 and 31, wherein the linker is present in the MenA, MenC, MenW-135 or MenY conjugate at a ratio of one linker per 10-100 sugar repeating units.

[0038] Embodiment 33 is the vaccine composition of embodiments 30 and 31, wherein the linker is present in the MenA, MenC, MenW-135 or MenY conjugate at a ratio of one linker per 20-60 sugar repeating units.

[0039] Embodiment 34 is the vaccine composition of embodiments 30 and 31, wherein the linker comprises a spacer between the first carbonyl group and the second carbonyl group, and the spacer comprises 4-8 carbons.

[0040] Embodiment 35 is the vaccine composition of any one of embodiments 30-34, wherein the linker of the MenA conjugate comprises the residue of a dihydrazide.

[0041] Embodiment 36 is the vaccine composition of embodiment 35, wherein the linker of the MenA conjugate comprises the residue of adipic acid dihydrazide.

[0042] Embodiment 37 is the vaccine composition of any one of the preceding embodiments, wherein the polysaccharide of the MenA, MenC, MenW-135 and / or MenY conjugate is attached to the carrier protein via a linker of formula I: Wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein.

[0043] Embodiment 38 is the vaccine composition of any one of embodiments 30-37, wherein the linker is in the MenA conjugate.

[0044] Embodiment 39 is the vaccine composition of any one of embodiments 30-37, wherein the linker is in the MenC conjugate.

[0045] Embodiment 40 is the vaccine composition of any one of embodiments 30-37, wherein the linker is in the MenW-135 conjugate.

[0046] Embodiment 41 is the vaccine composition of any one of embodiments 30-37, wherein the linker is in the MenY conjugate.

[0047] Embodiment 42 is the vaccine composition of any one of the preceding embodiments, wherein the polysaccharide of the MenA, MenC, MenW-135 and / or MenY conjugate is attached to a carrier protein as shown in Formula II: PR-NH-CH 2 -PS(II), wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein.

[0048] Embodiment 43 is the vaccine composition of embodiment 42, wherein the polysaccharide of the MenA conjugate is attached to a carrier protein as shown in formula II.

[0049] Embodiment 44 is the vaccine composition of embodiment 42, wherein the polysaccharide of the MenC conjugate is attached to a carrier protein as shown in formula II.

[0050] Embodiment 45 is the vaccine composition of embodiment 42, wherein the polysaccharide of the MenW-135 conjugate is attached to a carrier protein as shown in formula II.

[0051] Embodiment 46 is the vaccine composition of embodiment 42, wherein the polysaccharide of the MenY conjugate is attached to a carrier protein as shown in formula II.

[0052] Embodiment 47 is the vaccine composition of any of the preceding embodiments, wherein the carrier protein comprises or consists of recombinant exoprotein A (rEPA), diphtheria toxoid or diphtheria toxin B-fragment, CRM197, tetanus toxoid or tetanus toxin C-fragment.

[0053] Embodiment 48 is the vaccine composition of any one of the preceding embodiments, wherein the carrier protein is tetanus toxoid.

[0054] Embodiment 49 is a method for producing a conjugate of a capsular polysaccharide of Neisseria meningitidis and a carrier protein, comprising: a) activating the polysaccharide with an activator that can form a carbamate linkage, wherein the activator is present in a 20-fold to 50-fold molar excess relative to the polysaccharide; b) partially quenching the activated polysaccharide and derivatizing the activated polysaccharide with a dihydrazide linker added at a molar ratio of 0.3 to 1.0 relative to the polysaccharide repeating units, wherein the polysaccharide is derivatized at a ratio of one dihydrazide linker per 10-100 sugar repeating units; c) conjugating the derivatized polysaccharide to the carrier protein by carbodiimide chemistry, wherein the polysaccharide is present at a weight ratio of 3:1 to 5:1 relative to the carrier protein at the beginning of the conjugation reaction, thereby forming the conjugate.

[0055] Embodiment 50 is the method of embodiment 49, wherein the dihydrazide linker is added at a molar ratio of 0.4 to 0.6 relative to the polysaccharide repeating units.

[0056] Embodiment 51 is the method of embodiment 49, comprising the further step of quenching the reaction with glycerol.

[0057] Embodiment 52 is the method of any one of embodiments 49 to 51, wherein the dihydrazide linker is adipic acid dihydrazide.

[0058] Embodiment 53 is the method of any one of embodiments 49 to 51, wherein the starting concentration of the derivatized polysaccharide in the conjugation reaction is 10 g / L to 20 g / L.

[0059] Embodiment 54 is the method of any one of embodiments 49 to 51, wherein the activator comprises a carbonyl group bound to two N-linked heteroaryl groups, such as CDI (1,1'-carbonyldiimidazole) and CDT (1,1'-carbonyl-di-(1,2,4-triazole)), or other suitable leaving groups.

[0060] Embodiment 55 is the method of embodiment 54, wherein the activating agent is carbonyldiimidazole.

[0061] Embodiment 56 is the method of any one of embodiments 49 to 51, wherein the activating agent is present in the activation step in a 35-fold to 45-fold molar excess relative to the polysaccharide.

[0062] Embodiment 57 is the method of any one of embodiments 49 to 51, wherein the conjugating step comprises reacting the carrier protein with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.

[0063] Embodiment 58 is the method of any one of embodiments 49 to 51, wherein the polysaccharide is a MenA capsular polysaccharide.

[0064] Embodiment 59 is the method of any one of embodiments 49 to 51, wherein the polysaccharide is a MenC capsular polysaccharide.

[0065] Embodiment 60 is the method of any one of embodiments 49 to 51, wherein the polysaccharide is MenW-135 or MenY capsular polysaccharide.

[0066] Embodiment 61 is a method for producing a conjugate of a capsular polysaccharide of Neisseria meningitidis and a carrier protein, comprising: a) partially de-O-acetylation of the polysaccharide by alkaline hydrolysis; b) activating the polysaccharide by periodate treatment, thereby converting the diol into an aldehyde to the extent of at least 20 nmol aldehyde per mg of polysaccharide; c) conjugating the activated polysaccharide to the carrier protein by reductive amination, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1:1 to 5:1 relative to the carrier protein, thereby forming the conjugate.

[0067] Embodiment 62 is the method of embodiment 61, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1.5 to 3:1 relative to the carrier protein.

[0068] Embodiment 63 is the method of embodiment 61, wherein the de-O-acetylation reduces the initial amount of O-acetylation in the polysaccharide by 40% to 70%, or 50% to 60%.

[0069] Embodiment 64 is the method of any one of embodiments 61 to 63, wherein after de-O-acetylation, the degree of O-acetylation of the polysaccharide is from 0.6 μmol / mg polysaccharide to 1.5 μmol / mg polysaccharide or from 0.8 to 1.4 μmol / mg polysaccharide.

[0070] Embodiment 65 is the method of embodiment 64, wherein the degree of O-acetylation is greater than or equal to 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2 μmol / mg polysaccharide.

[0071] Embodiment 66 is the method of embodiment 64, wherein the degree of O-acetylation is less than or equal to 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 or 1.4 μmol / mg polysaccharide.

[0072] Embodiment 67 is the method of any one of embodiments 61 to 66, wherein the initial concentration of the activated polysaccharide in the conjugation reaction is 20 g / L to 50 g / L.

[0073] Embodiment 68 is the method of any one of embodiments 61 to 66, wherein the polysaccharide is a MenC capsular polysaccharide.

[0074] Embodiment 69 is the method of any one of embodiments 61 to 66, wherein the polysaccharide is a MenA capsular polysaccharide.

[0075] Embodiment 70 is the method of any one of embodiments 61 to 66, wherein the polysaccharide is MenW-135 or MenY capsular polysaccharide.

[0076] Embodiment 71 is the method of any one of embodiments 61 to 66, wherein the size of the polysaccharide is reduced to 30 to 150 kDa or 50 to 100 kDa prior to the conjugation reaction.

[0077] Embodiment 72 is a method for producing a conjugate of a capsular polysaccharide of Neisseria meningitidis and a carrier protein, comprising: a) activating the polysaccharide by treatment with periodate, thereby converting the diol into an aldehyde to the extent of at least 50 nmol aldehyde per mg of polysaccharide; b) conjugating the activated polysaccharide to the carrier protein by reductive amination, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1:1 to 5:1 relative to the carrier protein, thereby forming the conjugate.

[0078] Embodiment 73 is the method of embodiment 72, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1.5 to 3:1 relative to the carrier protein.

[0079] Embodiment 74 is the method of any one of embodiments 72 and 73, wherein the polysaccharide is MenW-135 or MenY capsular polysaccharide.

[0080] Embodiment 75 is the method of any one of embodiments 72 and 73, wherein the polysaccharide is a MenC capsular polysaccharide.

[0081] Embodiment 76 is the method of any one of embodiments 72 and 73, wherein the polysaccharide is a MenA capsular polysaccharide.

[0082] Embodiment 77 is the method of any one of embodiments 72 to 76, wherein the size of the polysaccharide is reduced to 100 to 200 kDa or 125 to 175 kDa prior to the conjugation reaction.

[0083] Embodiment 78 is the method of any one of embodiments 49, 61 and 72, wherein the size of the polysaccharide is reduced by acid hydrolysis and / or heating.

[0084] Embodiment 79 is the method of any one of embodiments 49, 61 and 72, wherein the size of the polysaccharide is reduced by oxidative cleavage.

[0085] Embodiment 80 is the method of any one of embodiments 72 to 79, wherein the reductive amination comprises the use of cyanoborohydride or other reducing agents such as pyridine borane (C 5 H 8 BN) and methylpyridine borane complex (C 6 H7 N·BH 3 ) reduces the imine to the amine.

[0086] Embodiment 81 is the method of any one of embodiments 72 to 79, further comprising converting unreacted aldehyde in the conjugate to an alcohol with a reducing agent.

[0087] Embodiment 82 is the method of embodiment 81, wherein the reducing agent is a borohydride.

[0088] Embodiment 83 is the method of any one of embodiments 72 to 79, wherein periodate is added to a concentration of 1 mM to 4 mM or 1.5 mM to 3 mM to activate the polysaccharide.

[0089] Embodiment 84 is the method of any one of embodiments 49 to 83, further comprising purifying the conjugate by hydrophobic interaction chromatography.

[0090] Embodiment 85 is the method of any one of embodiments 49 to 83, further comprising purifying the conjugate by mixed mode resin chromatography.

[0091] Embodiment 86 is a method for purifying a conjugate of a capsular polysaccharide of Neisseria meningitidis and a carrier protein from a mixture containing the conjugate, salt and free polysaccharide, comprising: a) contacting a hydrophobic interaction chromatography resin with the mixture, wherein the conjugate binds to the resin; b) eluting the free polysaccharide from the resin; and c) eluting the conjugate from the resin with an aqueous liquid, wherein the aqueous liquid does not contain salt or contains less salt than the mixture, thereby obtaining a composition comprising the purified conjugate.

[0092] Embodiment 87 is the method of embodiment 86, wherein the salt comprises ammonium sulfate.

[0093] Embodiment 88 is the method of embodiment 86 or embodiment 87, wherein the mixture comprises the salt in an amount ranging from 0.5 to 1.5M or 0.8 to 1.2M.

[0094] Embodiment 89 is the method of any one of embodiments 86 to 88, wherein the aqueous liquid comprises less than 0.2, 0.1 or 0.05 M salt.

[0095] Embodiment 90 is the method of any one of embodiments 86 to 89, wherein the aqueous liquid is water.

[0096] Embodiment 91 is the method of any one of embodiments 86 to 90, wherein the composition comprises less than 20% by weight free polysaccharides, less than 10% by weight free polysaccharides, less than 5% by weight free polysaccharides, or is substantially free of free polysaccharides.

[0097] Embodiment 92 is the method of any one of embodiments 86 to 91, wherein the hydrophobic interaction chromatography resin is a phenyl, propyl or butyl resin.

[0098] Embodiment 93 is the method of any one of embodiments 49 to 92, wherein the carrier protein is tetanus toxoid.

[0099] Embodiment 94 is a conjugate produced according to the method of any one of embodiments 49 to 85.

[0100] Embodiment 95 is a vaccine composition comprising: a) a first conjugate of MenA capsular polysaccharide and a carrier protein; b) a second conjugate of MenC capsular polysaccharide and a carrier protein; c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein one, two, three or all of the first, second, third and fourth conjugates are produced according to the method of any one of embodiments 49 to 85.

[0101] Embodiment 96 is the vaccine composition of any one of embodiments 1-48 or 95, which is free of an adjuvant.

[0102] Embodiment 97 is the vaccine composition of any one of embodiments 1-48 or 95-96, further comprising a pharmaceutically acceptable buffer.

[0103] Embodiment 98 is the vaccine composition of embodiment 97, comprising acetate buffer having a pH of 5.5 to 6.5.

[0104] Embodiment 99 is the vaccine composition of any one of embodiments 1-48 or 95-98, further comprising a pharmaceutically acceptable salt.

[0105] Embodiment 100 is the vaccine composition of embodiment 99, wherein the pharmaceutically acceptable salt is sodium chloride.

[0106] Embodiment 101 is the vaccine composition of embodiment 99 or 100, wherein the pharmaceutically acceptable salt is present at 0.45% w / v to 0.9% w / v, or 0.5% w / v to 0.85% w / v.

[0107] Embodiment 102 is the vaccine composition of any one of embodiments 1-48 or 95-101, wherein at least one, two, three or all four of the first, second, third and fourth conjugates comprise multiple attachment points between the polysaccharide and the carrier protein.

[0108] Embodiment 103 is the vaccine composition of any one of embodiments 1-48 or 95-102, formulated for intramuscular administration.

[0109] Embodiment 104 is a single unit dose of the vaccine composition of any one of embodiments 1-48 or 95-103 comprising 6 μg to 15 μg of each of the MenA, MenC, MenW-135 and MenY polysaccharides.

[0110] Embodiment 105 is the single unit dose of embodiment 104, wherein the carrier protein is present in an amount of 50 μg to 80 μg.

[0111] Embodiment 106 is a single unit dose of embodiment 104 or 105 contained in a syringe.

[0112] Embodiment 107 is the single unit dose of embodiment 106, wherein the syringe is free of silicone.

[0113] Embodiment 108 is the single unit dose of embodiment 106 or 107, wherein the syringe is packaged for commercial sale or distribution.

[0114] Embodiment 109 is a method of vaccinating a subject against Neisseria meningitidis, comprising administering to the subject a dose of the vaccine composition of any one of embodiments 1-48 or 95-103. Embodiment 109A is the vaccine composition of any one of embodiments 1-48 or 95-103 for use in a method of vaccinating a subject against Neisseria meningitidis, comprising administering to the subject a dose of the vaccine composition.

[0115] Embodiment 110 is the use of the vaccine composition of any one of embodiments 1-48 or 95-103 or a single unit dose of any one of embodiments 104-108 for immunizing a subject against Neisseria meningitidis. Embodiment 110A is the vaccine composition of any one of embodiments 1-48 or 95-103 or a single unit dose of any one of embodiments 104-108 for immunizing a subject against Neisseria meningitidis.

[0116] Embodiment 111 is the use of a vaccine composition of any one of embodiments 1-48 or 95-103 or a single unit dose of any one of embodiments 104-108 for the manufacture of a medicament for immunizing a subject against Neisseria meningitidis. Embodiment 111A is a vaccine composition of any one of embodiments 1-48 or 95-103 or a single unit dose of any one of embodiments 104-108 for use as a medicament for immunizing a subject against Neisseria meningitidis.

[0117] Embodiment 112 is the method, use, or composition of any one of embodiments 109-111A, wherein the vaccine is administered intramuscularly.

[0118] Embodiment 113 is the method, use, or composition of any one of embodiments 109-112, wherein the subject is between 6 weeks and 3 years old.

[0119] Embodiment 114 is the method, use or composition of embodiment 113, wherein the subject is 2 months old, 6 months old, 12 months old or 15 months old.

[0120] Embodiment 115 is the method, use, or composition of any one of embodiments 109-112, wherein the subject is 50 years or older, 55 years or older, 60 years or older, or 65 years or older.

[0121] Embodiment 116 is the method, use or composition of any one of embodiments 109-115, wherein the vaccine is administered as a 0.5 mL dose.

[0122] Embodiment 117 is the method, use or composition of embodiment 116, wherein the vaccine comprises 4-10 μg of each of serogroups A, C, Y and W-135.

[0123] Embodiment 118 is the method, use or composition of embodiment 116, wherein the vaccine comprises 50-80 μg of tetanus toxoid protein.

[0124] Embodiment 119 is the method, use or composition of embodiment 116, wherein the vaccine comprises 4-10 μg of each of serogroups A, C, Y and W-135, and 50-80 μg of tetanus toxoid protein.

[0125] Embodiment 120 is the method, use or composition of any one of embodiments 109-119, further comprising administering a vaccine not directed against Neisseria meningitidis simultaneously with, but not in the same injection as, the MenACYW-TT vaccine.

[0126] Embodiment 121 is the method, use or composition of embodiment 120, wherein the non-meningitidis Neisseria vaccine is for preventing varicella, diphtheria, Hib, hepatitis B, measles, mumps, pertussis, polio, pneumococcal, rotavirus, rubella or tetanus infection.

[0127] Embodiment 122 is the method, use or composition of embodiment 120, wherein the non-meningitidis Neisseria vaccine is DTaP5, Hib, HepB, DTap5-IPV / Hib, DTap5-IPV / Hib, HepB, MMR, IPV, PCV7, PCV13, RV1 or RV5.

[0128] Embodiment 123 is the method, use, or composition of any one of embodiments 109-122, wherein the subject has previously received a Neisseria meningitidis capsular saccharide conjugate vaccine.

[0129] Embodiment 124 is the method, use or composition of embodiment 123, wherein the subject received the Neisseria meningitidis capsular saccharide conjugate vaccine 4 months to 10 years ago. Embodiment 124A is the method, use or composition of embodiment 123, wherein the subject received the Neisseria meningitidis capsular saccharide conjugate vaccine 2 months to 10 years ago.

[0130] Embodiment 125 is the method, use, or composition of any one of embodiments 109-122, wherein the subject has not previously received a Neisseria meningitidis capsular saccharide conjugate vaccine.

[0131] Specifically, the present invention includes but is not limited to the following:

[0132] 1. A Neisseria meningitidis vaccine composition comprising:

[0133] a) a first conjugate of MenA capsular polysaccharide and a carrier protein;

[0134] b) a second conjugate of MenC capsular polysaccharide and a carrier protein;

[0135] c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and

[0136] d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein;

[0137] wherein the second conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide both attached to the carrier protein via a secondary amine, and the O-acetylation level of the polysaccharide of the second conjugate is 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide.

[0138] 2. A Neisseria meningitidis vaccine composition comprising:

[0139] a) a first conjugate of MenA capsular polysaccharide and a carrier protein;

[0140] b) a second conjugate of MenC capsular polysaccharide and a carrier protein;

[0141] c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and

[0142] d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein;

[0143] wherein the second conjugate is a population comprising a single-end-linked conjugated polysaccharide attached to the carrier protein via a secondary amine, wherein the single-end-linked conjugated polysaccharide has a terminal unlinked sugar, wherein the terminal sugar has a primary hydroxyl or secondary amine linkage at position 7, or wherein the reducing end is modified with a (2-hydroxy)ethoxy or secondary amine linkage.

[0144] 3. A Neisseria meningitidis vaccine composition comprising:

[0145] a) a first conjugate of MenA capsular polysaccharide and a carrier protein;

[0146] b) a second conjugate of MenC capsular polysaccharide and a carrier protein;

[0147] c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and

[0148] d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein;

[0149] wherein the MenA capsular polysaccharide is attached to the carrier protein via a linker comprising a carbamate, a spacer and an amide, wherein the spacer is between the carbamate and the amide and comprises 2-10 linear carbons, and the polysaccharide to carrier protein mass ratio of the first conjugate is 0.3 to 1.5.

[0150] 4. A Neisseria meningitidis vaccine composition comprising:

[0151] a) a first conjugate of MenA capsular polysaccharide and a carrier protein;

[0152] b) a second conjugate of MenC capsular polysaccharide and a carrier protein;

[0153] c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and

[0154] d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein;

[0155] wherein the MenA capsular polysaccharide is attached to the carrier protein via a linker comprising a carbamate, a spacer and an amide, wherein the spacer is between the carbamate and the amide and comprises 2-10 linear carbons; and wherein the MenC, MenW-135 and MenY capsular polysaccharides are attached to the carrier protein via secondary amines;

[0156] And at least one of the conjugates has a weight average molecular weight ranging from 300 kDa to 1500 kDa.

[0157] 5. A Neisseria meningitidis vaccine composition comprising:

[0158] a) a first conjugate of MenA capsular polysaccharide and a carrier protein;

[0159] b) a second conjugate of MenC capsular polysaccharide and a carrier protein;

[0160] c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and

[0161] d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein;

[0162] wherein the carrier protein is tetanus toxoid;

[0163] The weight average molecular weight of one or more of the first, second, third and fourth conjugates ranges from 300 kDa to 1500 kDa; and the composition comprises less than 20% by weight of free polysaccharide relative to total polysaccharide.

[0164] 6. A Neisseria meningitidis vaccine composition comprising:

[0165] a) a first conjugate of MenA capsular polysaccharide and a carrier protein;

[0166] b) a second conjugate of MenC capsular polysaccharide and a carrier protein;

[0167] c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and

[0168] d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein;

[0169] wherein the carrier protein is tetanus toxoid;

[0170] The polysaccharide to carrier protein mass ratio of one or more of the first, second, third and fourth conjugates is 0.3 to 1.5; and the composition comprises less than 20% by weight of free polysaccharide relative to total polysaccharide.

[0171] 7. The vaccine composition of any of the preceding items, wherein the first, second, third and / or fourth conjugate is a population comprising molecules with a molecular weight ranging from 700 kDa to 1400 kDa or from 800 kDa to 1300 kDa.

[0172] 8. A Neisseria meningitidis vaccine composition comprising a conjugate of MenC capsular polysaccharide and a carrier protein, wherein the conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide both attached to the carrier protein via a secondary amine, and the O-acetylation level of the polysaccharide of the conjugate of MenC capsular polysaccharide and the carrier protein ranges from 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide.

[0173] 9. The vaccine composition of claim 8, wherein the conjugate

[0174] a) having a weight average molecular weight ranging from 300 kDa to 1500 kDa; or

[0175] b) is a population comprising molecules with a molecular weight in the range of 700 kDa to 1400 kDa or 800 kDa to 1300 kDa.

[0176] 10. The vaccine composition of any one of items 4, 5, 7 or 9, wherein the molecular weight is determined by multi-angle light scattering (MALS).

[0177] 11. The vaccine composition of any of the preceding items, wherein the degree of O-acetylation of the MenC polysaccharide ranges from 0.6 to 1.5 μmol / mg polysaccharide or from 0.8 to 1.4 μmol / mg polysaccharide.

[0178] 12. The vaccine composition of claim 11, wherein the degree of O-acetylation is greater than or equal to 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2 μmol / mg polysaccharide.

[0179] 13. The vaccine composition of claim 11, wherein the degree of O-acetylation is less than or equal to 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 or 1.4 μmol / mg polysaccharide.

[0180] 14. The vaccine composition of any of the preceding items, wherein the conjugate comprising MenC polysaccharide is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide.

[0181] 15. The vaccine composition of claim 14, wherein the single-end-linked polysaccharide of the second conjugate comprises a terminally unlinked sugar, wherein the single-end-linked conjugated polysaccharide has a terminally unlinked sugar, wherein the terminal sugar has a primary hydroxyl group at position 7, or wherein the reducing end is modified by a (2-hydroxy)ethoxy group.

[0182] 16. The vaccine composition of any of the preceding items, wherein the conjugate comprising MenC polysaccharide comprises one or more modifications selected from the group consisting of: (i) a primary hydroxyl group at position 7, (ii) a (2-hydroxy)ethoxy group at the reducing end, and (iii) conjugation to the carrier protein, wherein the modification is present at no less than 25 nmol / mg polysaccharide.

[0183] 17. A vaccine composition according to any preceding item, comprising a conjugate of MenW-135 and / or MenY polysaccharide comprising one or more modifications selected from: (i) primary hydroxyl groups at vicinal diol positions in native MenW-135 or MenY polysaccharide and (ii) conjugation to a carrier protein, wherein the modifications are present at not less than 60 nmol / mg polysaccharide.

[0184] 18. The vaccine composition of claim 16 or 17, wherein the modification is present in an amount of less than 200 nmol / mg polysaccharide, less than 150 nmol / mg polysaccharide, less than 150 nmol / mg polysaccharide, less than 100 nmol / mg polysaccharide or less than 80 nmol / mg polysaccharide.

[0185] 19. The vaccine composition of any preceding item, wherein the size of the MenC polysaccharide is reduced by 3-8 times relative to the native MenC polysaccharide.

[0186] 20. The vaccine composition of any of the preceding items, comprising a conjugate of MenA capsular polysaccharide and a carrier protein, the polysaccharide to carrier protein mass ratio of the conjugate being from 0.5 to 1.5 or from 0.7 to 1.4.

[0187] 21. The vaccine composition of claim 20, wherein the polysaccharide to carrier protein mass ratio of the MenA conjugate is 0.8 to 1.3.

[0188] 22. The vaccine composition of any preceding item, comprising a conjugate of MenC and / or MenY capsular polysaccharide and a carrier protein, the conjugate having a polysaccharide to carrier protein mass ratio of 0.3 to 1.1.

[0189] 23. The vaccine composition of claim 22, wherein the polysaccharide to carrier protein mass ratio of the MenC conjugate is 0.4 to 0.8.

[0190] 24. The vaccine composition of any one of the preceding items, comprising a conjugate of MenW-135 capsular polysaccharide and a carrier protein, the polysaccharide to carrier protein mass ratio of the conjugate being 0.3 to 1.3.

[0191] 25. The vaccine composition of claim 24, wherein the polysaccharide to carrier protein mass ratio of the MenW-135 conjugate is 0.6 to 1.3.

[0192] 26. The vaccine composition of any of the preceding items, comprising a conjugate of MenY capsular polysaccharide and a carrier protein, the polysaccharide to carrier protein mass ratio of the conjugate being between 0.5 and 1.3.

[0193] 27. The vaccine composition of claim 26, wherein the polysaccharide to carrier protein mass ratio of the MenY conjugate is 0.5 to 0.9.

[0194] 28. The vaccine composition of any of the preceding items, wherein the composition comprises less than 20% by weight of free polysaccharides.

[0195] 29. The vaccine composition of claim 28, wherein the composition comprises less than 10% by weight of free polysaccharides, less than 5% by weight of free polysaccharides, or is substantially free of free polysaccharides.

[0196] 30. The vaccine composition of any preceding item, wherein the polysaccharide of the MenA, MenC, MenW-135 or MenY conjugate is attached to the carrier protein via a linker.

[0197] 31. The vaccine composition of claim 30, wherein the linker comprises 2-10 linear carbons.

[0198] 32. The vaccine composition of items 30 and 31, wherein the linker is present in the MenA, MenC, MenW-135 or MenY conjugate at a ratio of one linker per 10-100 sugar repeating units.

[0199] 33. The vaccine composition of items 30 and 31, wherein the linker is present in the MenA, MenC, MenW-135 or MenY conjugate at a ratio of one linker per 20-60 sugar repeating units.

[0200] 34. The vaccine composition of items 30 and 31, wherein the linker comprises a spacer between the first carbonyl group and the second carbonyl group, and the spacer comprises 4-8 carbons.

[0201] 35. The vaccine composition of any one of items 30-34, wherein the linker of the MenA conjugate comprises the residue of a dihydrazide.

[0202] 36. The vaccine composition of claim 35, wherein the linker of the MenA conjugate comprises the residue of adipic acid dihydrazide.

[0203] 37. The vaccine composition of any of the preceding items, wherein the polysaccharide of the MenA, MenC, MenW-135 and / or MenY conjugate is attached to the carrier protein via a linker of formula I:

[0204]

[0205] Wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein.

[0206] 38. The vaccine composition of any one of items 30-37, wherein the linker is in the MenA conjugate.

[0207] 39. The vaccine composition of any one of items 30-37, wherein the linker is in the MenC conjugate.

[0208] 40. The vaccine composition of any one of items 30-37, wherein the linker is in the MenW-135 conjugate.

[0209] 41. The vaccine composition of any one of items 30-37, wherein the linker is in the MenY conjugate.

[0210] 42. The vaccine composition of any of the preceding items, wherein the polysaccharide of the MenA, MenC, MenW-135 and / or MenY conjugate is attached to a carrier protein as shown in formula II:

[0211] PR-NH-CH 2 -PS(II)

[0212] Wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein.

[0213] 43. The vaccine composition of claim 42, wherein the polysaccharide of the MenA conjugate is attached to a carrier protein as shown in formula II.

[0214] 44. The vaccine composition of claim 42, wherein the polysaccharide of the MenC conjugate is attached to a carrier protein as shown in formula II.

[0215] 45. The vaccine composition of claim 42, wherein the polysaccharide of the MenW-135 conjugate is attached to a carrier protein as shown in formula II.

[0216] 46. ​​The vaccine composition of claim 42, wherein the polysaccharide of the MenY conjugate is attached to a carrier protein as shown in formula II.

[0217] 47. The vaccine composition of any of the preceding items, wherein the carrier protein comprises or consists of recombinant exoprotein A (rEPA), diphtheria toxoid or diphtheria toxin B-fragment, CRM197, tetanus toxoid or tetanus toxin C-fragment.

[0218] 48. The vaccine composition of any of the preceding items, wherein the carrier protein is tetanus toxoid.

[0219] 49. A method for producing a conjugate of Neisseria meningitidis capsular polysaccharide and a carrier protein, comprising:

[0220] a) activating the polysaccharide with an activator capable of forming carbamate linkages, wherein the activator is present in a 20-fold to 50-fold molar excess relative to the polysaccharide;

[0221] b) partially quenching the activated polysaccharide and derivatizing the activated polysaccharide with a dihydrazide linker added at a molar ratio of 0.3 to 1.0 relative to the polysaccharide repeating units, wherein the polysaccharide is derivatized at a ratio of one dihydrazide linker per 10-100 saccharide repeating units;

[0222] c) conjugating the derivatized polysaccharide to the carrier protein by carbodiimide chemistry, wherein the polysaccharide is present in a weight ratio of 3:1 to 5:1 relative to the carrier protein at the start of the conjugation reaction, thereby forming the conjugate.

[0223] 50. The method of claim 49, wherein the dihydrazide linker is added at a molar ratio of 0.4 to 0.6 relative to the polysaccharide repeating units.

[0224] 51. The method of claim 49, comprising the further step of quenching the reaction with glycerol.

[0225] 52. The method of any one of items 49 to 51, wherein the dihydrazide linker is adipic acid dihydrazide.

[0226] 53. The method of any one of items 49 to 51, wherein the initial concentration of the derivatized polysaccharide in the conjugation reaction is 10 g / L to 20 g / L.

[0227] 54. The method of any one of items 49 to 51, wherein the activator comprises a carbonyl group bound to two N-linked heteroaryl groups, such as CDI (1,1'-carbonyldiimidazole) and CDT (1,1'-carbonyl-di-(1,2,4-triazole)), or other suitable leaving groups.

[0228] 55. The method of claim 54, wherein the activator is carbonyldiimidazole.

[0229] 56. The method of any one of items 49 to 51, wherein the activating agent is present in the activation step in a 35-fold to 45-fold molar excess relative to the polysaccharide.

[0230] 57. The method of any one of items 49 to 51, wherein the conjugation step comprises reacting the carrier protein with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.

[0231] 58. The method of any one of items 49 to 51, wherein the polysaccharide is a MenA capsular polysaccharide.

[0232] 59. The method of any one of items 49 to 51, wherein the polysaccharide is a MenC capsular polysaccharide.

[0233] 60. The method of any one of items 49 to 51, wherein the polysaccharide is MenW-135 or MenY capsular polysaccharide.

[0234] 61. A method for producing a conjugate of Neisseria meningitidis capsular polysaccharide and a carrier protein, comprising:

[0235] a) partially de-O-acetylation of the polysaccharide by alkaline hydrolysis;

[0236] b) activating the polysaccharide by treatment with periodate to convert the diol to an aldehyde to an extent of at least 20 nmol aldehyde per mg of polysaccharide;

[0237] c) conjugating the activated polysaccharide to the carrier protein by reductive amination, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1:1 to 5:1 relative to the carrier protein, thereby forming the conjugate.

[0238] 62. The method of claim 61, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1.5 to 3:1 relative to the carrier protein.

[0239] 63. The method of claim 61, wherein the de-O-acetylation reduces the initial amount of O-acetylation in the polysaccharide by 40% to 70%, or 50% to 60%.

[0240] 64. The method of any one of items 61 to 63, wherein after de-O-acetylation, the degree of O-acetylation of the polysaccharide is 0.6 μmol / mg polysaccharide to 1.5 μmol / mg polysaccharide or 0.8 to 1.4 μmol / mg polysaccharide.

[0241] 65. The method of claim 64, wherein the degree of O-acetylation is greater than or equal to 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2 μmol / mg polysaccharide.

[0242] 66. The method of claim 64, wherein the degree of O-acetylation is less than or equal to 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 or 1.4 μmol / mg polysaccharide.

[0243] 67. The method of any one of items 61 to 66, wherein the initial concentration of the activated polysaccharide in the conjugation reaction is 20 g / L to 50 g / L.

[0244] 68. The method of any one of items 61 to 66, wherein the polysaccharide is a MenC capsular polysaccharide.

[0245] 69. The method of any one of items 61 to 66, wherein the polysaccharide is a MenA capsular polysaccharide.

[0246] 70. The method of any one of items 61 to 66, wherein the polysaccharide is MenW-135 or MenY capsular polysaccharide.

[0247] 71. The method of any one of items 61 to 66, wherein the size of the polysaccharide is reduced to 30 to 150 kDa or 50 to 100 kDa prior to the conjugation reaction.

[0248] 72. A method for producing a conjugate of Neisseria meningitidis capsular polysaccharide and a carrier protein, comprising:

[0249] a) activating the polysaccharide by treatment with periodate to convert the diol to an aldehyde to an extent of at least 50 nmol aldehyde per mg of polysaccharide;

[0250] b) conjugating the activated polysaccharide to the carrier protein by reductive amination, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1:1 to 5:1 relative to the carrier protein, thereby forming the conjugate.

[0251] 73. The method of claim 72, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1.5 to 3:1 relative to the carrier protein.

[0252] 74. The method of any one of items 72 and 73, wherein the polysaccharide is MenW-135 or MenY capsular polysaccharide.

[0253] 75. The method of any one of items 72 and 73, wherein the polysaccharide is a MenC capsular polysaccharide.

[0254] 76. The method of any one of items 72 and 73, wherein the polysaccharide is a MenA capsular polysaccharide.

[0255] 77. The method of any one of items 72 to 76, wherein the size of the polysaccharide is reduced to 100 to 200 kDa or 125 to 175 kDa prior to the conjugation reaction.

[0256] 78. The method of any one of items 49, 61 and 72, wherein the size of the polysaccharide is reduced by acid hydrolysis and / or heating.

[0257] 79. The method of any one of items 49, 61 and 72, wherein the size of the polysaccharide is reduced by oxidative cleavage.

[0258] 80. The method of any one of items 72 to 79, wherein the reductive amination comprises reducing the imine to the amine using cyanoborohydride or other reducing agents such as pyridine borane (C5H8BN) and methyl pyridine borane complex (C6H7N·BH3).

[0259] 81. The method of any one of items 72 to 79, further comprising converting unreacted aldehyde in the conjugate into an alcohol using a reducing agent.

[0260] 82. The method of claim 81 , wherein the reducing agent is a borohydride.

[0261] 83. The method of any one of items 72 to 79, wherein periodate is added to a concentration of 1 mM to 4 mM or 1.5 mM to 3 mM to activate the polysaccharide.

[0262] 84. The method of any one of items 49 to 83, further comprising purifying the conjugate by hydrophobic interaction chromatography.

[0263] 85. The method of any one of items 49 to 83, further comprising purifying the conjugate by mixed mode resin chromatography.

[0264] 86. A method for purifying a conjugate of a capsular polysaccharide of Neisseria meningitidis and a carrier protein from a mixture containing the conjugate, salt and free polysaccharide, comprising:

[0265] a) contacting a hydrophobic interaction chromatography resin with the mixture, wherein the conjugate is bound to the resin;

[0266] b) eluting free polysaccharides from the resin; and

[0267] c) eluting the conjugate from the resin with an aqueous liquid, wherein the aqueous liquid contains no salt or contains less salt than the mixture, thereby obtaining a composition comprising a purified conjugate.

[0268] 87. The method of claim 86, wherein the salt comprises ammonium sulfate.

[0269] 88. The method of claim 86 or claim 87, wherein the mixture comprises salt in an amount ranging from 0.5 to 1.5 M or from 0.8 to 1.2 M.

[0270] 89. The method of any one of items 86 to 88, wherein the aqueous liquid comprises less than 0.2, 0.1 or 0.05 M salt.

[0271] 90. The method of any one of items 86 to 89, wherein the aqueous liquid is water.

[0272] 91. The method of any one of items 86 to 90, wherein the composition comprises less than 20% by weight free polysaccharides, less than 10% by weight free polysaccharides, less than 5% by weight free polysaccharides, or is substantially free of free polysaccharides.

[0273] 92. The method of any one of items 86 to 91, wherein the hydrophobic interaction chromatography resin is a phenyl, propyl or butyl resin.

[0274] 93. The method of any one of items 49 to 92, wherein the carrier protein is tetanus toxoid.

[0275] 94. A conjugate produced according to the method of any one of items 49 to 85.

[0276] 95. A vaccine composition comprising:

[0277] a) a first conjugate of MenA capsular polysaccharide and a carrier protein;

[0278] b) a second conjugate of MenC capsular polysaccharide and a carrier protein;

[0279] c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and

[0280] d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein;

[0281] Wherein one, two, three or all of the first, second, third and fourth conjugates are produced according to the method of any one of items 49 to 85.

[0282] 96. The vaccine composition of any one of items 1-48 or 95, which does not contain an adjuvant.

[0283] 97. The vaccine composition of any one of items 1-48 or 95-96, further comprising a pharmaceutically acceptable buffer.

[0284] 98. The vaccine composition of claim 97, comprising an acetate buffer having a pH of 5.5 to 6.5.

[0285] 99. The vaccine composition of any one of items 1-48 or 95-98, further comprising a pharmaceutically acceptable salt.

[0286] 100. The vaccine composition of claim 99, wherein the pharmaceutically acceptable salt is sodium chloride.

[0287] 101. The vaccine composition of item 99 or 100, wherein the pharmaceutically acceptable salt is present at 0.45% w / v to 0.9% w / v, or 0.5% w / v to 0.85% w / v.

[0288] 102. The vaccine composition of any one of items 1-48 or 95-101, wherein at least one, two, three or all four of the first, second, third and fourth conjugates comprise multiple attachment points between the polysaccharide and the carrier protein.

[0289] 103. The vaccine composition of any one of items 1-48 or 95-102, which is formulated for intramuscular administration.

[0290] 104. A single unit dose of the vaccine composition of any one of items 1-48 or 95-103 comprising 6 μg to 15 μg of each of the MenA, MenC, MenW-135 and MenY polysaccharides.

[0291] 105. A single unit dose of claim 104, wherein the carrier protein is present in an amount of 50 μg to 80 μg.

[0292] 106. A single unit dose of item 104 or 105 contained in a syringe.

[0293] 107. The single unit dose of claim 106, wherein the syringe is free of silicone.

[0294] 108. The single unit dose of item 106 or 107, wherein the syringe is packaged for commercial sale or distribution.

[0295] 109. A method of vaccinating a subject against Neisseria meningitidis, comprising administering to the subject a dose of the vaccine composition of any one of items 1-48 or 95-103.

[0296] 110. Use of the vaccine composition of any one of items 1-48 or 95-103 or a single unit dose of any one of items 104-108 for immunizing a subject against Neisseria meningitidis.

[0297] 111. Use of the vaccine composition of any one of items 1-48 or 95-103 or a single unit dose of any one of items 104-108 for the manufacture of a medicament for immunizing a subject against Neisseria meningitidis.

[0298] 112. The method or use of any one of items 109-111, wherein the vaccine is administered intramuscularly.

[0299] 113. The method or use of any one of items 109-112, wherein the subject is 6 weeks to 3 years old.

[0300] 114. The method of claim 113, wherein the subject is 2 months old, 6 months old, 12 months old or 15 months old.

[0301] 115. The method or use of any one of items 109-112, wherein the subject is 50 years or older, 55 years or older, 60 years or older, or 65 years or older.

[0302] 116. The method or use of any one of items 109-115, wherein the vaccine is administered in a 0.5 mL dose.

[0303] 117. The method or use of claim 116, wherein the vaccine comprises 4-10 μg of each of serogroups A, C, Y and W-135.

[0304] 118. The method or use of claim 116, wherein the vaccine comprises 50-80 μg of tetanus toxoid protein.

[0305] 119. The method or use of claim 116, wherein the vaccine comprises 4-10 μg of each of serogroups A, C, Y and W-135, and 50-80 μg of tetanus toxoid protein.

[0306] 120. The method or use of any one of items 109 to 119, further comprising administering a vaccine not directed against Neisseria meningitidis simultaneously with, but not in the same injection as, the MenACYW-TT vaccine.

[0307] 121. The method or use of claim 120, wherein the non-meningitidis Neisseria vaccine is used to prevent varicella, diphtheria, Hib, hepatitis B, measles, mumps, pertussis, poliomyelitis, pneumococcal, rotavirus, rubella or tetanus infection.

[0308] 122. The method or use of claim 120, wherein the non-meningitidis Neisseria vaccine is DTaP5, Hib, HepB, DTap5-IPV / Hib, DTap5-IPV / Hib, HepB, MMR, IPV, PCV7, PCV13, RV1 or RV5.

[0309] 123. The method or use of any one of items 109-122, wherein the subject has previously received a Neisseria meningitidis capsular saccharide conjugate vaccine.

[0310] 124. The method or use of claim 123, wherein the subject received a Neisseria meningitidis capsular saccharide conjugate vaccine 4 months to 10 years ago.

[0311] 125. The method or use of any one of items 109-122, wherein the subject has not previously received a Neisseria meningitidis capsular saccharide conjugate vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0312] Figure 1A A schematic diagram of a serogroup A polysaccharide-ADH-linked protein conjugate is shown. Serogroup A polysaccharide 10 with reactive site residue 11 is formed by carbonyldiimidazole (CDI) activation and optionally derivatization with adipic acid dihydrazide (ADH) on the hydroxyl groups of the polysaccharide and reacting with a protein (e.g., tetanus toxoid (TT)). The activated / derivatized polysaccharide is cross-linked to protein 13 at group 14 via linkage 12, either directly or indirectly. For example, direct linkage can use primary amines of the protein, such as by forming a carbamate linkage (e.g., derived from CDI). Indirect linkage can be derived from ADH and N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDAC), which activates the carboxyl groups of the protein.

[0313] Figure 1B The preparation of an active O-acylisourea intermediate of a carrier protein (e.g., TT) is shown, using N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDAC) to react with a carboxyl group of the protein (e.g., at an aspartic acid or glutamic acid side chain or the C-terminus). This intermediate is suitable for coupling to an amine group of an activated derivatized polysaccharide (not shown).

[0314] Figure 1CA general scheme for producing activated derivatized serogroup A polysaccharides is shown, which can be used to produce serogroup A polysaccharide-ADH conjugates linked to carrier proteins (e.g., TT). In this embodiment, the polysaccharide is activated by CDI at the hydroxyl groups to form an imidazole carbamate reactive intermediate, which is further derivatized with ADH. The ADH-derivatized serogroup A polysaccharide is suitable for covalent attachment to a carrier protein via amine coupling of the primary amine group on the ADH linker with the reactive O-acylisourea intermediate of the carrier protein (not shown).

[0315] Figure 1D A general scheme for producing a conjugate of a serogroup A polysaccharide linked to tetanus toxoid via a carbamate is shown. The polysaccharide (PS) is activated by CDI at the hydroxyl groups to form an imidazole carbamate reactive intermediate. The reactive intermediate is then reacted with a protein carrier (PR). The carbamate linkage is formed by a nucleophilic substitution reaction in which a primary amine of the protein attacks the carbamate carbon, resulting in the loss of imidazole and the formation of a carbamate linkage between the polysaccharide and the protein.

[0316] Figure 1E -F shows the structure of serogroup A polysaccharide after (E) CDI activation and (F) CDI activation and derivatization with ADH.

[0317] Figure 1G Shown is the production of a serogroup A polysaccharide-ADH conjugate linked to a carrier protein (e.g., TT) from an activated derivatized polysaccharide and an active O-acylisourea intermediate formed by a tetanus toxoid carrier protein and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDAC). The primary amine of the activated derivatized polysaccharide replaces the isourea, which acts as a leaving group, to give a product in which the protein is linked to the polysaccharide via an amide bond, the residue of ADH, and a carbamate connection, wherein the carbonyl is derived from CDI. The eliminated urea byproduct is not shown.

[0318] Figure 1H Shows Figure 1F The product of the reaction pulls out the structure of the linked polysaccharide repeating units (including ADH residues).

[0319] Figure 2A A schematic diagram of a serogroup C polysaccharide-protein conjugate is shown. A serogroup C polysaccharide 20 is bound to a protein (eg, tetanus toxoid) 21 at its terminus.

[0320] Figure 2B Activation of serogroup C polysaccharide using sodium metaperiodate is shown (shown with conventional carbon numbering in the polysaccharide repeat unit). Sodium metaperiodate treatment results in cleavage between carbons 7 and 8, oxidatively depolymerizing the polysaccharide to the corresponding terminal aldehyde.

[0321] Figure 2CThe formation of a serogroup C polysaccharide-protein (e.g., TT) conjugate by reductive amination is shown. A primary amine (e.g., a lysine side chain or the N-terminus) of a protein (PR) reacts with the terminal aldehyde of a depolymerized activated serogroup C polysaccharide (PS) to form a Schiff base intermediate (not shown), which is reduced (e.g., using pyridine borane, methyl pyridine borane, or cyanoborohydride) to afford a secondary amine linkage. The polysaccharide moiety is linked to the protein terminus. A single protein molecule may react with more than one polysaccharide, and some polysaccharide termini may be unreacted (not shown; see Figure 2A ). Following Schiff base reduction (not shown), the unreacted aldehyde can be capped, ie, reduced to an alcohol, using a suitable reducing agent such as sodium borohydride.

[0322] Figure 2D Shows Figure 2C The product of the reaction in the middle pulls out the structure of linked polysaccharide repeating units. Linking of proteins to other polysaccharides is possible (not shown).

[0323] Figure 3 A schematic diagram of a serogroup W-135 or serogroup Y polysaccharide-protein conjugate is shown. A serogroup W-135 or serogroup Y polysaccharide 31 is conjugated to one or more proteins (e.g., tetanus toxoid) 30 at one or more locations.

[0324] Figure 4A Depolymerization and activation of serogroup W-135 polysaccharide is shown. The polysaccharide is depolymerized using, for example, elevated temperature and then activated by treatment with sodium metaperiodate, which cleaves vicinal diols (such as, for example, between carbons 7 and 8 of the sialic acid moiety) and oxidizes them to aldehydes.

[0325] Figure 4B The formation of a serogroup W-135 polysaccharide-protein (e.g., TT) conjugate by reductive amination is shown. The primary amine (e.g., lysine side chain or N-terminus) of the protein (PR) reacts with the aldehyde of the depolymerized activated serogroup W-135 polysaccharide (PS) to form a Schiff base intermediate (not shown). Reduction of the intermediate by sodium cyanoborohydride affords a secondary amine linkage. A single protein molecule can react with more than one polysaccharide, and vice versa (not shown; see Figure 3 ). Following Schiff base reduction (not shown), the unreacted aldehyde can be capped, ie, reduced to an alcohol, using a suitable reducing agent such as sodium borohydride.

[0326] Figure 4C Shows Figure 4B The product of the reaction in , pulls out one possible structure of linked polysaccharide repeating units. Linking of proteins to other polysaccharides (or vice versa) is possible (not shown; see Figure 3 ).

[0327] Figure 5A Depolymerization and activation of serogroup Y polysaccharide is shown. The polysaccharide is depolymerized using, for example, elevated temperature, and then activated by treatment with sodium metaperiodate, which cleaves vicinal diols, such as, for example, between carbons 7 and 8 of the sialic acid moiety, and oxidizes them to aldehydes.

[0328] Figure 5B The formation of a serogroup Y polysaccharide-protein (e.g., TT) conjugate by reductive amination is shown. A primary amine (e.g., a lysine side chain or the N-terminus) of a protein (PR) reacts with the aldehyde of a depolymerized activated serogroup Y polysaccharide (PS) to form a Schiff base intermediate (not shown). Reduction of this intermediate (e.g., using pyridine borane, methyl pyridine borane, or cyanoborohydride) affords a secondary amine linkage. A single protein molecule can react with more than one polysaccharide, and vice versa (not shown; see Figure 3 ). Following Schiff base reduction (not shown), the unreacted aldehyde can be capped, ie, reduced to an alcohol, using a suitable reducing agent such as sodium borohydride.

[0329] Figure 5C Shows Figure 5B The product of the reaction in , pulls out one possible structure of linked polysaccharide repeating units. Linking of proteins to other polysaccharides (or vice versa) is possible (not shown; see Figure 3 ).

[0330] Figure 6 The percentage of subjects in Groups 1-4 who achieved hSBA titers greater than or equal to 1:8 for serogroups A, C, Y, and W 30 days after the last vaccine dose (D30) is shown.

[0331] Figure 7 Shown are the percentages of subjects in Groups 1-4 who achieved rSBA titers greater than or equal to 1:8 for serogroups A, C, Y, and W on D30 after the last vaccine dose.

[0332] Figure 8 The cumulative percentage of participants reporting one or more solicited injection site reactions within 7 days after administration of the MenACYW-TT vaccine is shown.

[0333] Fig. 9 Shown are the percentages of subjects with solicited systemic reactions within 7 days of administration of MenACYW-TT vaccine plus conventional vaccine or conventional vaccine alone.

[0334] Fig.10 showed that the use of MenACYW-TT or - Percentage of subjects achieving hSBA titers ≥ 1:8 for different serogroups on D30 post-A / C / Y / W-135 (MPSV4) vaccination.

[0335] Fig.11 Shown is a summary of hSBA geometric mean titers (GMTs) at D30 following administration of MenACYW-TT or MPSV4 vaccines.

[0336] Fig.12 Shown are the percentages of subjects achieving rSBA titers ≥ 1:8 for the different serogroups at D30 after administration of MenACYW-TT or MPSV4 vaccine.

[0337] Fig.13 Shown are the percentages of subjects with solicited injection site reactions by type and grade following administration of MenACYW-TT or MPSV4 vaccine.

[0338] Fig.14 Shown are the percentages of subjects with solicited systemic reactions by type and grade following administration of MenACYW-TT or MPSV4 vaccine.

[0339] Fig.15 Shown are the percentages of subjects achieving hSBA vaccine serological responses at D30 after administration of MenACYW-TT or MCV4-TT vaccines.

[0340] Fig.16 Shown are the geometric means of post-vaccination hSBA following administration of MenACYW-TT or MCV4-TT vaccines.

[0341] Fig.17 Shown are solicited injection site reactions from D0-D7 after administration of MenACYW-TT or MCV4-TT vaccines.

[0342] Fig.18 Solicited systemic reactions from D0-D7 after administration of MenACYW-TT or MCV4-TT vaccines are shown. DETAILED DESCRIPTION OF THE INVENTION

[0343] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Although the present invention will be described in conjunction with the enumerated embodiments, it should be understood that they are not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents that may be included in the present invention defined by the appended claims.

[0344] Before describing the present teachings in detail, it should be understood that the disclosure is not limited to specific compositions or process steps, as these may vary. It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates, and reference to "a cell" includes a plurality of cells, etc.

[0345] Numerical ranges include the numbers defining the range. Measured values ​​and measurable values ​​are understood to be approximate, taking into account significant figures and errors associated with the measurements. Moreover, the use of "comprising," "containing," and "including" is not limiting. It should be understood that the foregoing general description and detailed description are merely exemplary and explanatory and are not intended to limit the present teachings.

[0346] Unless otherwise specified in the above description, embodiments described in this specification as “comprising” various components are also considered to be “consisting of (the components)” or “consisting essentially of (the components)”; embodiments described in this specification as “consisting of (the components)” are also considered to be “comprising” the components or “consisting essentially of (the components)”; and embodiments described in this specification as “consisting essentially of (the components)” are also considered to be “consisting of (the components)” or “comprising” the components (this interchangeability does not apply to the use of these terms in the claims).

[0347] The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. If any document incorporated by reference conflicts with any term defined in this specification, the present specification shall prevail. Although the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings are limited to these embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teachings encompass various alternatives, modifications, and equivalents.

[0348] A. Definition

[0349] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings:

[0350] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the terms listed before the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items / terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that, unless otherwise apparent from the context, there is typically no limit to the number of items / terms in any combination.

[0351] As used herein, the term "kit" refers to a packaged set of related components, such as one or more compounds or compositions and one or more related materials, such as solvents, solutions, buffers, instructions, or desiccants.

[0352] Unless the context requires otherwise, "or" is used in its inclusive sense, ie, equivalent to "and / or".

[0353] The terms "linker" and "linkage" are used interchangeably and refer to a chemical moiety comprising a chain of atoms that covalently attaches or is attached to an item, such as a carrier protein or polysaccharide.

[0354] "Linking moiety" means a chemically reactive group, substituent, or moiety, eg, a nucleophile or electrophile, capable of reacting with another molecule to form a linkage via a covalent bond.

[0355] "Alkyl" refers to a saturated or unsaturated branched, straight, branched or cyclic hydrocarbon group derived from a single carbon atom of a parent alkane, alkene or alkyne by removing a hydrogen atom. Typical alkyl groups are composed of 1 to 12 saturated and / or unsaturated carbons, including but not limited to methyl, ethyl, propyl, butyl, etc.

[0356] A "repeating unit" is a monosaccharide or oligosaccharide residue that is polymerized in a polysaccharide. The repeating units of MenA and MenC are monosaccharides (N-acetylmannosamine and sialic acid, respectively), and the repeating units of MenW-135 and MenY are disaccharides (sialic acid and glucose for MenY, or sialic acid and galactose for MenW-135). The repeating unit may vary from one to another with respect to side chains (e.g., O-acetylation) and / or modifications (such as those disclosed herein).

[0357] MenA, MenC, MenW-135 and MenY are used as abbreviations for serogroup A, C, W-135 or Y Neisseria meningitidis or its capsular polysaccharide, respectively (as in the case of e.g. "MenC conjugate", which means a conjugate of the capsular polysaccharide of serogroup C Neisseria meningitidis with a carrier protein).

[0358] B. Exemplary Vaccine Compositions

[0359] In some embodiments, vaccine compositions are provided. In some embodiments, the vaccine composition comprises a conjugate of MenC capsular polysaccharide and a carrier protein. In some embodiments, the vaccine composition comprises a conjugate of MenA capsular polysaccharide and a carrier protein. In some embodiments, the vaccine composition comprises a conjugate of MenW-135 capsular polysaccharide and a carrier protein. In some embodiments, the vaccine composition comprises a conjugate of MenY capsular polysaccharide and a carrier protein. In some embodiments, the vaccine composition comprises at least two, at least three, or at least four conjugates of capsular polysaccharide and a carrier protein. The at least two, at least three, or at least four conjugates of capsular polysaccharide and a carrier protein can be conjugates of capsular polysaccharides from different Neisseria meningitidis serogroups, for example, conjugates of MenA, MenC, MenY, and MenW capsular polysaccharides and a carrier protein, such as tetanus toxoid.

[0360] Capsular polysaccharides can be prepared according to the method described in US2003 / 0068336 Example 1. Capsular polysaccharides can also be prepared using the culture medium and method described in, for example, US 6,933,137.

[0361] Conjugates comprising carrier proteins are disclosed herein. Examples of protein carriers are discussed below, for example, Pichichero ME. Protein carriers of conjugate vaccines: Characteristics, development, and clinical trials. Human Vaccines & Immunotherapeutics. 2013; 9(12): 2505-2523. doi: 10.4161 / hv.26109, which is incorporated herein by reference. In some embodiments, the carrier protein comprises or consists of recombinant exoprotein alpha (REPA), diphtheria toxoid, CRM197, tetanus toxoid, or tetanus toxin C-fragment. In some embodiments, the protein carrier is tetanus toxoid.

[0362] In some embodiments, tetanus toxoid (TT) is prepared by extraction, ammonium sulfate purification, and formalin inactivation of toxin from a culture of Clostridium tetani (Harvard strain) grown in Mueller and Miller medium or modified Mueller and Miller medium. In some embodiments, TT is treated to reduce residual formaldehyde, concentrated in sodium chloride, and filter sterilized. In some embodiments, TT is purified by chromatography rather than ammonium sulfate purification. In some embodiments, modified Mueller and Miller medium does not contain bovine heart infusion. In some embodiments, Clostridium tetani is grown in the medium described in Table 3, page 16 of WO 2006 / 042542.

[0363] Certain embodiments discussed below relate to features, such as chemical moieties (e.g., hydroxyl groups or O-acetylation) or conjugation to a carrier protein, that are present in a given amount per unit mass of polysaccharide. For example, a feature or combination of features may be present at a level of not less than 25 nmol / mg of polysaccharide. This means that in 1 mg of polysaccharide, the feature or combination of features occurs at least 15 x 10 15 times (where 25 nmol = 25 x 10 -9 mole x (6.02 x 10 23 terms / mole) = 15 x 10 15 item).

[0364] In some embodiments, the O-acetylation level of the polysaccharide of the conjugate of MenC capsular polysaccharide and carrier protein ranges from 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide. In some embodiments, the O-acetylation level is greater than or equal to 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2 μmol / mg polysaccharide. In some embodiments, the O-acetylation level is less than or equal to 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 μmol / mg polysaccharide. For example, the level can be 0.6 to 1.5 μmol / mg polysaccharide or 0.8 to 1.4 μmol / mg polysaccharide. The O-acetyl content can be measured by the Hestrin method (Hestrin et al., J. Biol. Chem. 1949, 180, p. 249).

[0365] In some embodiments, in the composition, the weight average molecular weight of at least one of the conjugates ranges from 300 kDa to 1500 kDa. In some such embodiments, the weight average molecular weight is greater than or equal to 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, or 1100 kDa. In some such embodiments, the weight average molecular weight is less than or equal to 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa or 1400 kDa. The weight average molecular weight can be determined by methods known in the art, such as multi-angle light scattering (MALS). In some embodiments, at least one conjugate in the composition comprises (i.e., as a population of molecules comprising the following) molecules with a molecular weight in the range of 700 kDa to 1400 kDa. It should be noted that regardless of whether the weight average molecular weight or number average molecular weight is within the range, some molecules in the population may have weights within the range. For example, a population of molecules with a weight average molecular weight of 600 kDa or 1500 kDa may contain molecules with a molecular weight in the range of 700 kDa to 1400 kDa. In some embodiments, at least one conjugate in the composition comprises a molecule with a molecular weight in the range of 800 kDa to 1300 kDa. In some embodiments, at least one conjugate in the composition comprises a molecule with a molecular weight in the range of 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, or 1400-1500 kDa. In some embodiments, the MenA conjugate has a molecular weight as described above. In some embodiments, the MenC conjugate has a molecular weight as described above. In some embodiments, the MenW-135 conjugate has a molecular weight as described above. In some embodiments, the MenY conjugate has a molecular weight as described above. In some embodiments, the molecular weight is determined using multi-angle light scattering (MALS). In some embodiments, the molecular weight is determined using high performance size exclusion chromatography (HPSEC).

[0366] In some embodiments, the conjugate (e.g., a conjugate of MenC) is a population comprising a single-end-linked conjugated polysaccharide, a double-end-linked polysaccharide, or a combination thereof. A single-end-linked conjugated polysaccharide is attached to a carrier protein at one end. A double-end-linked conjugated polysaccharide is attached to a carrier protein at both ends. For example, an end-linked conjugated polysaccharide can be formed by cleaving and activating a vicinal diol in the polysaccharide backbone to expose an activated end, such as by periodate treatment. For example, the MenC polysaccharide has a 7,8 vicinal diol in its sialic acid repeat unit to the extent that positions 7 and 8 are not O-acetylated in the same repeat unit. The vicinal diol is in the backbone of the polysaccharide because cleaving it separates one sugar from the next, i.e., the vicinal diol is not part of a side chain. After activation, the activated end can then react with a carrier protein (or react with a linker, which will then be attached to the carrier protein or has already been attached to the carrier protein) to form an end-linked polysaccharide conjugate. If only one end (terminal sugar residue) of the polysaccharide is linked to the carrier protein (including through a linker if applicable), the conjugate is single-end linked. Double-end linked conjugates have carrier proteins linked to both ends of the polysaccharide. Generally, using a higher stoichiometric amount of polysaccharide or a lower total reactant concentration relative to the carrier protein will bias the conjugation reaction toward a single-end linked product. Conversely, a lower stoichiometric amount of polysaccharide or a higher total reactant concentration relative to the carrier protein will bias the conjugation reaction toward a double-end linked product.

[0367] In some embodiments, a single end linked conjugated polysaccharide (e.g., a MenC conjugate) has a terminal unlinked sugar with a primary hydroxyl group at position 7, or wherein the reducing end is modified with a (2-hydroxy)ethoxy group. This can be produced by activating a polysaccharide containing a 7,8 vicinal diol with periodate (which gives a terminal aldehyde), conjugating at one end, and reducing the unreacted aldehyde at the other end (e.g., with a borohydride reagent). The primary hydroxyl group at position 7 can be considered to be one end of a truncated sialic acid residue. A reducing end modified with a (2-hydroxy)ethoxy group can be considered to be a fragment of a sialic acid residue attached at its reducing end to the 9th and 8th carbons and their associated oxygens of another residue.

[0368] In some embodiments, the conjugate is a MenW-135 and / or MenY polysaccharide comprising one or more modifications selected from: (i) a primary hydroxyl group at a vicinal diol position in a native MenW-135 or MenY polysaccharide and (ii) conjugation to the carrier protein, wherein the modification is present in no less than 60 nmol / mg polysaccharide. The modification may be formed by periodate oxidation followed by conjugation to a carrier protein and reduction of the unreacted aldehyde. Periodate-driven cleavage of sugar residues may occur at vicinal diol positions, such as positions 7, 8 or 8, 9 of sialic acid, and may also occur in the hexose ring of the repeating unit, particularly where the diol is in a cis arrangement. In some embodiments, the modification is present in an amount of less than 200 nmol / mg polysaccharide, less than 150 nmol / mg polysaccharide, less than 150 nmol / mg polysaccharide, less than 100 nmol / mg polysaccharide, or less than 80 nmol / mg polysaccharide.

[0369] In some embodiments, the polysaccharide is linked to the carrier protein via a secondary amine. In some embodiments, the polysaccharide is attached to the carrier protein as shown in Formula II:

[0370] PR-NH-CH 2 -PS(II)

[0371] Wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein. Such secondary amine linkages can be formed, for example, by reductive amination, in which a primary amine on a protein (e.g., the N-terminus or amino group of a lysine side chain) attacks an activated group (e.g., an aldehyde) on a polysaccharide to form a Schiff base, which is then reduced to form a secondary amine. Reduction can be performed using a suitable reducing agent, such as a cyanoborohydride (e.g., sodium cyanoborohydride) or a borane (e.g., pyridine borane or methyl pyridine borane).

[0372] In some embodiments, the size of the conjugate of MenC polysaccharide is reduced by 3-8 times, e.g., 3-4 times, 4-5 times, 5-6 times, 6-7 times, or 7-8 times, relative to native MenC polysaccharide. Periodate cleavage separates adjacent repeat units and thereby reduces the size of the polysaccharide. Further size reduction may be achieved by treatment such as heating and / or acid (e.g., prior to periodate treatment). Other treatments known for size reduction, such as sonication or microfluidization, may also be used.

[0373] In some embodiments, the conjugate of MenA polysaccharide has a polysaccharide to carrier protein mass ratio of 0.3 to 1.5, such as 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, or 1.4 to 1.5. In some embodiments, the conjugate of MenA polysaccharide has a polysaccharide to carrier protein mass ratio of 0.5 to 1.5.

[0374] In some embodiments, the conjugate of MenC polysaccharide has a polysaccharide to carrier protein mass ratio of 0.3 to 1.1, such as 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0 or 1.0 to 1.1.

[0375] In some embodiments, the conjugate of MenW-135 polysaccharide has a polysaccharide to carrier protein mass ratio of 0.3 to 1.3, such as 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, or 1.2 to 1.3.

[0376] In some embodiments, the conjugate of MenY polysaccharide has a polysaccharide to carrier protein mass ratio of 0.5 to 1.3, such as 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, 0.9 to 1.0, 1.0 to 1.1, 1.1 to 1.2, or 1.2 to 1.3.

[0377] In some embodiments, the vaccine compositions provided herein contain less than 20% free polysaccharide by weight, e.g., less than 10% free polysaccharide by weight, less than 5% free polysaccharide by weight, or are substantially free of free polysaccharide. "Substantially free of free polysaccharide" means that the level of free polysaccharide is below the detection limit of a deoxycholate precipitation assay, in which a protein conjugated polysaccharide is precipitated with deoxycholate and the polysaccharide remaining in solution is determined, e.g., as described in Lei et al., "Quantification of free polysaccharide in meningococcal polysaccharide-diphtheria toxoid conjugate vaccines," Dev Biol (Basel). 2000; 103: 259-64 (PMID: 11214246).

[0378] In some embodiments, the polysaccharide is attached to the carrier protein by a linker comprising 2-10 straight-chain carbons (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons). "Straight-chain carbons" are carbons along the chain from the polysaccharide to the carrier protein, and do not include carbons on the branches of the chain. In some embodiments, the linker is included in a spacer between the first carbonyl and the second carbonyl, and the spacer comprises 4-8 carbons (e.g., 4, 5, 6, 7, or 8 carbons), which can be straight-chain carbons. The first carbonyl can be part of a carbamate. The second carbonyl can be part of an amide. The first carbonyl can be close to the polysaccharide and away from the carrier protein. The second carbonyl can be close to the carrier protein and away from the polysaccharide. The linker can include the residue of a dihydrazide, such as adipic acid dihydrazide (ADH). In some embodiments, the polysaccharide is attached to the carrier protein by a linker of formula I:

[0379]

[0380] Wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein. A single polysaccharide may be attached to one or more than one carrier protein (at different positions) and vice versa.

[0381] In some embodiments, the linker is present in the conjugate at a ratio of one linker per 10-100 (e.g., 20-60) saccharide repeating units. This includes linkers that are attached to both the carrier protein and the polysaccharide as well as linkers that are only attached to the polysaccharide, i.e., linkers that do not form an attachment to the carrier protein.

[0382] In some embodiments, a conjugate of a Neisseria meningitidis capsular polysaccharide with a carrier protein via a linker is provided, wherein the linker is present in an amount of 1 linker per 10-100 polysaccharide repeating units, such as 1 linker per 10-20 repeating units, 1 linker per 20-30 repeating units, 1 linker per 30-40 repeating units, 1 linker per 40-50 repeating units, 1 linker per 50-60 repeating units, or 1 linker per 60-70 repeating units. In some embodiments, the MenA polysaccharide is attached to the carrier protein via a linker as described above. In some embodiments, the MenC polysaccharide is attached to the carrier protein via a linker as described above.

[0383] In some embodiments, the polysaccharide conjugate compositions according to the present disclosure have improved stability relative to existing formulations. In some embodiments, stability is tested based on whether the free polysaccharide level corresponding to each conjugated polysaccharide remains below 40% after storage at 2°C-8°C for a period of time (e.g., 2.5, 3, 3.5, 4, or 4.5 years). In some embodiments, stability is tested based on whether the free polysaccharide level corresponding to each conjugated polysaccharide remains below 40% after storage at 23°C-27°C for a period of time (e.g., 2, 3, 4, 5, or 6 months). Certain tetravalent MenACYW polysaccharide conjugate vaccines require freeze-drying or other preservation measures, at least in part due to the low stability of one or more component conjugates as liquid formulations. Relative to a single liquid formulation, freeze-drying complicates both manufacturing and administration. In some embodiments, the polysaccharide is attached to the carrier protein at multiple points. Multiple points of attachment are typically the result of a conjugation chemistry, such as periodate activation followed by reductive amination, or carbonyldiimidazole-based coupling (optionally with a linker that can form a lattice of carrier protein and polysaccharide) and an appropriate polysaccharide size and loading ratio. For a detailed discussion of such exemplary chemistries, see the Examples below. Exemplary polysaccharide sizes and loading ratios compatible with the formation of a protein-polysaccharide lattice involving multiple attachment points are at least 30 kDa (and the exemplary size ranges discussed above) and a polysaccharide / protein ratio of 0.3 to 1.5 (and the exemplary loading ratio ranges discussed above). Without wishing to be bound by a particular theory, providing a conjugate having multiple attachment points between the polysaccharide and the carrier protein can contribute to the long-term stability of the conjugate, as multiple cleavage (e.g., hydrolysis) events will be required to release the polysaccharide fragments from the carrier protein. This contribution to long-term stability may be particularly relevant to MenA polysaccharides having phosphodiester linkages, which may be less stable than glycosidic bonds during storage in liquid. In some embodiments, the composition comprises a MenA polysaccharide having multiple attachment points to the carrier protein. In some embodiments, the composition comprises a MenC polysaccharide having multiple points of attachment to a carrier protein. In some embodiments, the composition comprises a MenY polysaccharide having multiple points of attachment to a carrier protein. In some embodiments, the composition comprises a MenW-135 polysaccharide having multiple points of attachment to a carrier protein. In some embodiments, the composition comprises a MenA, MenC, MenY, and MenW-135 polysaccharide, each having multiple points of attachment to a carrier protein.

[0384] In some embodiments, the vaccine compositions described herein are provided as liquid formulations in syringes (e.g., pre-filled and / or silicone-free syringes). In some embodiments, such syringes are commercially packaged for sale and / or distribution.

[0385] C. Exemplary Methods for Producing Conjugates and Vaccines

[0386] Provided herein are methods for producing and / or purifying conjugates of capsular polysaccharides and carrier proteins.

[0387] A reagent (such as an activating agent) is considered to be present in a given amount in a step (eg, an activation step) if it is present in such amount at any time during the relevant step or reaction (eg, when the reaction is initiated).

[0388] In some embodiments, a method for producing a conjugate of a polysaccharide with a carrier protein is provided. In some embodiments, the polysaccharide is de-O-acetylated, for example, by alkaline hydrolysis. For example, hydroxides such as NaOH or KOH can be used, for example, at a concentration of 50-150, 60-140, 70-130, 80-120, 90-110 or 95-100 mM. In some embodiments, the concentration of the hydroxide is 50, 60, 70, 80, 90 or 100 mM. In some embodiments, the concentration of the hydroxide is 100 mM. De-O-acetylation can be carried out at a time and temperature sufficient to de-O-acetylate the polysaccharide by 40% to 70% or 50% to 60%. In some embodiments, de-O-acetylation can be carried out at a time and temperature sufficient to de-O-acetylate the polysaccharide by 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the starting amount. In certain embodiments, after de-O-acetylation, the degree of O-acetylation of the polysaccharide is 0.6 μmol / mg polysaccharide to 1.5 μmol / mg polysaccharide or 0.8 to 1.4 μmol / mg polysaccharide. In some embodiments, the degree of O-acetylation is greater than or equal to 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 μmol / mg polysaccharide.

[0389] In some embodiments, the degree of O-acetylation is less than or equal to 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4 μmol / mg polysaccharide.

[0390] In some embodiments, the polysaccharide is depolymerized, for example, by heating, acid treatment, ultrasonic treatment, microfluidization, or a combination thereof (such as heating and acid). For example, the polysaccharide can be heated to 40°C-80°C (e.g., 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or 75°C) and / or exposed to a weakly acidic pH, such as 5, 5.5, 6, or 6.5 or higher. The polysaccharide can be depolymerized to a weight average molecular weight of, for example, 50 kDa to 200 kDa, such as 50, 75, 100, 125, 150, 175, or 200 kDa.

[0391] Some methods use activators that can form carbamate connections, examples of which are known in the art. The activators that can form carbamate connections can be compounds with a carbonyl group attached to two good leaving groups, such as heteroaryls connected by N-, such as imidazoles, pyridine, pyrimidine, purine, triazine, pyrazine, thiazine, thiazole, etc. In some embodiments, the activator is carbonyl diimidazole. The activator can be present in a molar excess of 20 to 50 times, for example 20 to 25 times, 25 to 30 times, 30 to 35 times, 35 to 40 times, 40 to 45 times, or 45 to 50 times relative to the polysaccharide.

[0392] In some embodiments, a linker such as a dihydrazide linker is reacted with an activated polysaccharide, for example, at a molar ratio of 0.3 to 1.0 relative to the polysaccharide repeating unit, for example, 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, or 0.9 to 1.0. In some embodiments, the dihydrazide linker is adipic acid dihydrazide. In some embodiments, the polysaccharide is derivatized with a linker so that it contains one linker per 10-100 repeating units, for example, one linker per 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 repeating units. In some embodiments, the polysaccharide is derivatized with a linker such that each of the 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 or 100 repeat units contain one linker.

[0393] In some embodiments, the polysaccharide is activated by treatment with an oxidizing agent such as a periodate, e.g., sodium metaperiodate (also known as sodium periodate). In some embodiments, the oxidizing agent reacts with the polysaccharide to form an aldehyde. In some embodiments, the aldehyde is formed on the side chain of the polysaccharide, e.g., for MenW-135 and MenY polysaccharides. In some embodiments, the aldehyde is formed by cleavage of the polysaccharide backbone and is formed at the termini, e.g., for MenC polysaccharides. The oxidation treatment may be performed using an oxidizing agent at around 2 mM, e.g., 1.5 to 3 mM. The oxidation treatment may be performed at 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 230 mM. The oxidation treatment can be carried out at a temperature and for a time sufficient to produce at least 20 nmol / mg polysaccharide, e.g., 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-160, 20-170, 20-180, 20-190 or 20-200 nmol / mg. The oxidation treatment can be carried out at a temperature and for a time sufficient to produce at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nmol / mg polysaccharide. In some embodiments, the oxidation treatment has a pH range of 6.5 to 9.5, or a pH of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5. In some embodiments, the oxidation treatment is performed in a liquid solution below room temperature, such as below 20°C, 15°C, 10°C, or 5°C.

[0394] When conjugation is performed by reductive amination, the Schiff base can be catalyzed by a suitable reducing agent such as cyanoborohydride (e.g., NaCNBH 3 ) or borane such as pyridine borane or methyl pyridine borane.

[0395] After conjugation, in the presence of unreacted aldehyde, the conjugate can be stabilized by reduction or amination of the aldehyde. 4 Ammonia, methylamine, glycine, alanine, etc. are suitable for amination of unreacted aldehydes.

[0396] In some embodiments, the conjugate is purified. A purification method involving ultrafiltration in the presence of ammonium sulfate is described in U.S. Patent No. 6,146,902. Alternatively, the conjugate can be purified from unreacted proteins and polysaccharides by many standard techniques, including size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, mixed mode resin chromatography or ammonium sulfate fractionation. See, for example, PW Anderson et al. (1986). J. Immunol. 137: 1181-1186. See also HJ Jennings and C. Lugowski (1981) J. Immunol. 127: 1011-1018.

[0397] In some embodiments, hydrophobic interaction chromatography (HIC) is performed. The following resins can be used, such as phenyl, hexyl, heptyl, octyl, nonyl or decyl resins. The mixture can be loaded onto the resin to purify the conjugate and separated from the free polysaccharide. In some embodiments, the mixture comprises a salt, such as ammonium sulfate. In some embodiments, the pH of the mixture being loaded is adjusted to make the pH closer to neutral. In some embodiments, the pH of the mixture being loaded is adjusted to 5.5-8.5, 6-8, 6.5 to 7.5 or 7. Salt can be present in a concentration of, for example, 0.5M to 1.5M, such as 0.5M to 0.7M, 0.7M to 0.9M, 0.9M to 1.1M, 1.1M to 1.3M or 1.3M to 1.5M. After loading, the resin can be washed with a saline solution, which, for example, comprises ammonium sulfate, wherein the salt concentration can be, for example, as shown in the previous sentence. In some embodiments, the resin is in a column, and the washing is at least two, three, four, five or six column volumes, for example, up to 7, 8, 9 or 10 column volumes. In some embodiments, the resin is washed in multiple batches of saline solutions, for example, two or more batches, wherein the volume of multiple batches of cumulative volumes is at least 2, 3, 4, 5, 6 or 7 times the volume of the mixture before loading. The conjugate can interact more strongly with the resin, for example, under high ionic strength, and free polysaccharides can be washed off. After washing with a low ionic strength eluent (such as water, such as WFI), the conjugate can be eluted from the resin. Alternatively, a low salt solution can be used, for example, a salt concentration (e.g., acetate, such as sodium acetate) is less than or equal to 0.2, 0.1, 0.05, 0.02, 0.01 or 0.005M. In some embodiments, the eluate comprises less than 20% by weight free polysaccharides, such as less than 10% by weight free polysaccharides, less than 5% by weight free polysaccharides, or is substantially free of free polysaccharides.

[0398] In some embodiments, a method of producing a conjugate of a capsular polysaccharide of Neisseria meningitidis and a carrier protein comprises

[0399] a) activating the polysaccharide with an activator capable of forming a carbamate linkage (e.g., carbonyldiimidazole), wherein the activator is present in a 20-fold to 50-fold molar excess relative to the polysaccharide;

[0400] b) quenching the activating agent (e.g., using water) and derivatizing the activated polysaccharide with a dihydrazide linker added at a molar ratio of 0.3 to 1.0 relative to polysaccharide repeating units, wherein the polysaccharide is derivatized at a ratio of one dihydrazide linker per 10-100 saccharide repeating units;

[0401] c) conjugating the derivatized polysaccharide to the carrier protein by carbodiimide chemistry, wherein the polysaccharide is present in a weight ratio of 3:1 to 5:1 relative to the carrier protein at the start of the conjugation reaction, thereby forming the conjugate.

[0402] In some embodiments, a method of producing a conjugate of a capsular polysaccharide of Neisseria meningitidis and a carrier protein comprises

[0403] a) partially de-O-acetylation of the polysaccharide by alkaline hydrolysis;

[0404] b) activating the polysaccharide by treatment with periodate to convert the diol to an aldehyde to an extent of at least 10, 15, 20, 25, 30, 35, 40 or 50 nmol aldehyde per mg of polysaccharide;

[0405] c) conjugating the activated polysaccharide to the carrier protein by reductive amination, wherein the polysaccharide is present in the conjugation reaction at a weight ratio of 0.5-1 to 5:1 relative to the carrier protein, thereby forming the conjugate.

[0406] In some embodiments, a method of producing a conjugate of a capsular polysaccharide of Neisseria meningitidis and a carrier protein comprises

[0407] a) activating the polysaccharide by periodate treatment to convert the diol to aldehyde to an extent of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 nmol aldehyde per mg of polysaccharide; and

[0408] b) conjugating the activated polysaccharide to the carrier protein by reductive amination, wherein the polysaccharide is present in the conjugation reaction in a weight ratio of 1:1 to 5:1 relative to the carrier protein, thereby forming the conjugate.

[0409] In some embodiments, the purification method further comprises purifying the conjugate by hydrophobic interaction chromatography (HIC).

[0410] A. Exemplary Vaccine Formulations

[0411] The preparation of the vaccine composition of the present invention can be accomplished using methods recognized in the art. The vaccine composition / preparation of the present invention may also contain one or more adjuvants. By way of example and not limitation, adjuvants include aluminum adjuvants, Freund's adjuvant, BAY, DC-chol, pcpp, monophosphoryl lipid A, CpG, QS-21, cholera toxin and formylmethionyl peptide. See, for example, Vaccine Design, the Subunit and Adjuvant Approach, 1995 (MF Powell and MJ Newman, ed., Plenum Press, NY). The adjuvant (if present) may be an aluminum adjuvant, such as aluminum hydroxide or aluminum phosphate. In some embodiments, vaccine compositions and preparations (e.g., MenACWY-TT vaccines) do not include adjuvants. In some embodiments, vaccine compositions and preparations (e.g., MenACWY-TT vaccines) include adjuvants.

[0412] The vaccine compositions and formulations of the invention (e.g., conjugate vaccines / MenACWY-TT vaccines) can be administered as a single dose or in a series (i.e., with one or more "booster doses"), or as a booster dose of a different N. meningitidis vaccine (e.g., a N. meningitidis capsular saccharide conjugate vaccine) administered earlier. For example, a child can receive a single dose early in life and then be given a booster dose up to 10 years later, as with other vaccines currently recommended for the prevention of childhood disease. In some embodiments, the vaccine doses described herein are administered 2 months to 10 years after a previous administration of a N. meningitidis capsular saccharide conjugate vaccine, such as 2 to 4 months, 4 to 6 months, 6 to 12 months, 1 to 2 years, 2 to 3 years, 3 to 4 years, 4 to 5 years, 5 to 6 years, 6 to 7 years, 7 to 8 years, 8 to 9 years, or 9 to 10 years after a previous administration of a N. meningitidis capsular saccharide conjugate vaccine.

[0413] The booster dose will generate antibodies from the primed B cells, i.e., an anamnestic response. That is, the vaccine compositions and formulations (e.g., MenACWY-TT vaccine) elicited higher initial (i.e., after a single administration of the vaccine) functional antibody responses in both young and elderly populations, and were able to elicit anamnestic responses (i.e., after a booster dose), demonstrating that the protective immune responses elicited by the vaccine compositions and formulations (e.g., MenACWY-TT vaccine) of the invention are long-lived.

[0414] In some embodiments, administration is intramuscular injection. In some embodiments, administration is subcutaneous, intradermal, intraperitoneal, parenteral or intravenous. Compositions and preparations can be mixed with suitable carriers, diluents or excipients, such as sodium acetate buffered saline solution, sterile water, normal saline, etc. Compositions / preparations can also be lyophilized. Compositions / preparations can contain auxiliary substances, such as wetting agents or emulsifiers, pH buffers, gelling or viscosity enhancing additives, preservatives, etc., depending on route of administration and desired preparations. Reference standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", the 17th edition, 1985 (incorporated herein by reference) prepare suitable preparations without excessive experiments.

[0415] In some embodiments, the vaccine composition / formulation is a liquid formulation. In some embodiments, the vaccine composition / formulation (e.g., MenACWY-TT vaccine) is a liquid composition that is administered by injection to animals, children (particularly small children), the elderly (e.g., over 55, 60, 65, 70, 75, 80, or 90).

[0416] The choice of appropriate carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form.

[0417] In one embodiment, vaccine compositions and formulations (e.g., MenACYW-TT vaccines) comprise pharmaceutically acceptable preservatives, carriers, buffers, excipients, etc. In one embodiment, pharmaceutically acceptable preservatives, carriers, or excipients increase or extend the shelf life of the composition. In some embodiments, the vaccine comprises a buffer. In some embodiments, the buffer is sodium acetate. In some embodiments, the buffer is sodium phosphate. In some embodiments, the buffer is present at a concentration ranging from 10mM to 100mM, for example, 10mM to 70mM, 15mM to 45mM, 20mM to 40mM, 40mM to 60mM, or 60mM to 100mM. In some embodiments, the pH of the buffer is 4.5 to 7.5, 4.5 to 7.0, 4.5 to 6.5, 4.5 to 6.0, 4.5 to 5.5, or 4.5 to 5.0. In some embodiments, the pH range of the buffer is 5.5 to 7.0, for example, 5.75 to 6.25 or 6.25 to 6.75. In some embodiments, the pH of the buffer is 5.5 to 6.5. In some embodiments, the pH of the buffer is 5 or 6. In some embodiments, the vaccine composition comprises a pharmaceutically acceptable salt. In some embodiments, the vaccine composition / formulation comprises saline. In some embodiments, the saline comprises NaCl or is NaCl. NaCl can be present in a concentration of 0.45% to 0.9% w / v, such as 0.5% to 0.85% w / v, or 0.6% to 0.8% w / v, or 0.6%, 0.67%, 0.75%, 0.8%, 0.85% or 0.9%.

[0418] In one embodiment, each component of the composition is chemically inert relative to the Neisseria meningitidis polysaccharide-protein carrier conjugate.

[0419] In some embodiments, vaccine compositions and formulations (e.g., MenACWY-TT vaccines) are formulated as a single unit dose. In some embodiments, a single unit dose comprises 6 μg to 15 μg of each of MenA, MenC, MenW-135, and MenY polysaccharides. In some embodiments, a single unit dose comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg of each of MenA, MenC, MenW-135, and MenY polysaccharides. In some embodiments, the carrier protein is present in an amount of 50 μg to 80 μg in a single unit dose. In some embodiments, the carrier protein is present in an amount of 45, 50, 55, 60, 65, 70, 75, or 80 μg in a single unit dose.

[0420] In some embodiments, vaccine compositions and formulations (e.g., MenACWY-TT vaccine) are formulated as a 0.5 mL dose in sodium acetate, sodium acetate buffered saline, or a similar buffer. In some embodiments, the 0.5 mL dose contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 μg of each of serogroups A, C, Y, and W-135 conjugated to 50, 55, 60, 65, 70, 80, 85, or 90 μg of tetanus toxoid protein. In some embodiments, the 0.5 mL dose is administered intramuscularly.

[0421] B. Exemplary Methods and Uses

[0422] In some embodiments, methods of vaccinating a subject against Neisseria meningitidis are contemplated, comprising administering the vaccine composition or formulation, e.g., the MenACWY-TT vaccine described herein. In some embodiments, the invention encompasses the use of a vaccine composition or formulation described herein for immunizing a subject against Neisseria meningitidis. In some embodiments, the use of a vaccine composition or formulation described herein for the preparation of a medicament for immunizing a subject against Neisseria meningitidis is contemplated.

[0423] In some embodiments, the subject is 12 months or older at the time of vaccination. In some embodiments, the subject is an elderly person at the time of vaccination. In some embodiments, the elderly person is 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 years old at the time of vaccination.

[0424] In some embodiments, the subject is 6 weeks to 3 years old at the time of vaccination. In some embodiments, the subject is 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks or 10 weeks at the time of vaccination. In some embodiments, the subject is 2 months, 4 months, 6 months, 12 months and / or 15 months at the time of vaccination. In some embodiments, the subject is vaccinated based on age. In some embodiments, three doses of a vaccine composition or formulation described herein (e.g., MenACWY-TT vaccine) are administered to the subject at 6-8, 10-12, and 14-16 weeks.

[0425] In some embodiments, the subject is vaccinated more than once in his lifetime. In some embodiments, the subject receives a booster dose 3 years or more after the first dose. In some embodiments, the subject receives a booster dose 4 years or more after the first dose. In some embodiments, the subject is vaccinated up to three times before his first birthday, and vaccinated once around or after his first birthday. In some embodiments, the first vaccination is at 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks of age. In some embodiments, the first or second vaccination is at 3, 4 or 5 months of age. In some embodiments, the first, second or third vaccination is at 5, 6 or 7 months of age. In some embodiments, the first, second, third or fourth vaccination is at 11, 12, 13, 14 or 15 months of age. In some embodiments, the first, second, third or fourth vaccination is at 14, 15, 16, 17 or 18 months of age. In some embodiments, the first vaccination is at 6, 7, 8 or 9 months, and the second vaccination is given at a maximum of 24 months. In some embodiments, the subject is vaccinated as an elderly person, regardless of whether they have previously received MenACWY-TT or other vaccines against Neisseria meningitidis. In some embodiments, the subject is 50 years of age or older, 55 years of age or older, 60 years of age or older, or 65 years of age or older when vaccinated with a vaccine composition or formulation described herein (e.g., MenACWY-TT vaccine).

[0426] In some embodiments, the vaccine compositions or formulations described herein (e.g., MenACWY-TT vaccine) are administered simultaneously with other conventional vaccines. In some embodiments, conventional vaccines include, for example, (DTaP5-IPV / Hib), (PCV7), Prevnar (PCV13), (RV5), (RV1), (HepB), RECOMBIVAX (HepB), (MMR), (MMR) and (V) Vaccines. In some embodiments, conventional vaccines include, for example, (Tdap5) and (HPV4). In some embodiments, conventional vaccines include DTaP5-IPV / HibHepB. Other conventional vaccines are known in the art and can be provided to the subject simultaneously with, before, or after the vaccine composition or formulation described herein (eg, MenACWY-TT vaccine). Example

[0427] The following are examples of methods, uses and compositions disclosed herein. It should be understood that various other embodiments can be implemented according to the general description and detailed description provided above. The following examples are given for the purpose of illustrating the present teaching content and should not be interpreted as limiting the scope of the present disclosure or claims.

[0428] 1. Preparation of Group A Conjugates

[0429] Example 1A

[0430] The purified capsular polysaccharide of Group A was dissolved in 10% by weight tetrabutylammonium chloride (TBAC) in dimethyl sulfoxide (DMSO) to a target concentration of 8 mg / mL. The solution was mixed until the polysaccharide was completely dissolved at 19°C-25°C. The dissolved polysaccharide was activated by adding a target concentration of 35-45 molar excess of carbonyldiimidazole (CDI) per N-acetylmannosamine phosphate repeating unit (PS RU) and mixed at 19°C-25°C for 50 to 70 minutes ( Figure 1C , first reaction; Figure 1E The polysaccharide solution was diluted 1:2 with WFI (50% v / v) to adjust the concentration of the activated polysaccharide to 4 mg / mL in 50% DMSO. The solution was derivatized by adding adipic acid dihydrazide (ADH) (1.0 mol ADH per 1-3 mol PS RU) ( Figure 1C , second reaction; Figure 1F The product shown in Figure 2) was added and mixed overnight at room temperature. The reaction resulted in an amount of derivatization such that there was one bound ADH per 10 to 100 polysaccharide repeating units, such as one bound ADH per 20, 30, 40, 50 or 60 polysaccharide repeating units. The activated polysaccharide was concentrated by ultrafiltration through a 10 kDa MWCO PES membrane and then diafiltered against 12-18 volumes of saline exchange. The target concentration was approximately 30 mg / mL. The activated polysaccharide was filtered and stored at 1°C-5°C.

[0431] Purified tetanus toxoid protein (TT) was filtered through a 0.2 micron membrane and stored at 1°C-5°C. The derivatized polysaccharide and concentrated tetanus protein were mixed together in a ratio of 0.5:1, 1:1, 2:1, 3:1, 4:1 or 5:1. An aliquot of 100 mg / mL of a cross-linking agent 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDAC) in 1.0 M MES buffer (pH 5.7) was added to the polysaccharide-protein mixture so that the final concentration of EDAC was 10 mg / mL and MES was 100 mM. Saline was added to obtain a target concentration of 16 mg / mL polysaccharide and 4 mg / mL TT. The final pH was adjusted to 5.5-5.9, and the reaction was mixed for 16-24 hours at 15.6°C-23.9°C. During this period, the EDAC and TT reacted to form an O-acylisourea intermediate ( Figure 1B Then, the O-acylisourea intermediate and the derivatized polysaccharide form a conjugate ( Figure 1G ; Figure 1H and Figure 1A The product shown in ).

[0432] Ammonium sulfate was added to the conjugation reaction to obtain a 1M ammonium sulfate concentration. The pH was adjusted to 7 and mixed until dissolved at room temperature. The conjugated reaction mixture was applied to a HIC column filled with phenyl resin. Unconjugated polysaccharides were eluted with 2 to 7 column volumes of 1M ammonium sulfate solution. The conjugate was eluted with WFI. In this embodiment and subsequent embodiments, the HIC purification of the conjugate can provide a product in which less than 20% of the polysaccharides by mass are free (unconjugated) polysaccharides. The conjugate eluate was diafiltered against 10 volumes of 50mM sodium acetate (pH 6.0) exchange using a 100kDa MWCO PES membrane. The purified conjugate was finally filtered using a 0.2 micron membrane and stored at 1°C-5°C.

[0433] Example 1B

[0434] The purified capsular polysaccharide of Group A was dissolved in tetrabutylammonium chloride (TBAC) / dimethyl sulfoxide (DMSO) (by weight) to a target concentration of 6 mg / mL. The solution was mixed at 19°C-25°C for 16 to 24 hours. The dissolved polysaccharide was activated by adding a target concentration of 35-45 molar excess of carbonyldiimidazole (CDI) per N-acetylmannosamine phosphate repeating unit (PS RU) and mixed at 19°C-25°C for 50 to 70 minutes ( Figure 1C , first reaction; Figure 1E The polysaccharide solution was diluted 1:2 with WFI (45%-50% v / v) such that the target concentration of the activated polysaccharide was 3 mg / mL in 50% DMSO.

[0435] Purified tetanus toxoid protein (TT) was filtered through a 0.2 micron membrane and stored at 1°C-5°C. Tetanus protein was added to a final concentration of 1 mg / mL. During this period, the activated polysaccharide and TT reacted to form a conjugate containing a carbamate linkage ( Figure 1D ). The reaction was allowed to proceed overnight at room temperature.

[0436] Ammonium sulfate was added to the conjugation reaction to obtain a 1M ammonium sulfate concentration. The pH was adjusted to 7 and mixed until dissolved at room temperature. The conjugated reaction mixture was applied to a HIC column filled with phenyl resin. Unconjugated polysaccharides were eluted with 2 to 7 column volumes of 1M ammonium sulfate solution. The conjugate was eluted with WFI. In this embodiment and subsequent embodiments, the HIC purification of the conjugate can provide a product in which less than 20% of the polysaccharides by mass are free (unconjugated) polysaccharides. The conjugate eluate was diafiltered against 10 volumes of 50mM sodium acetate (pH 6.0) exchange using a 100kDa MWCO PES membrane. The purified conjugate was finally filtered using a 0.2 micron membrane and stored at 1°C-5°C.

[0437] 2. Preparation of Group C Conjugate

[0438] Example 2A

[0439] The purified capsular polysaccharide of Group C was dissolved in normal saline to a target concentration of 10 mg / mL. The solution was mixed until dissolved. The temperature of the polysaccharide solution was adjusted to 37°C and sodium hydroxide (NaOH) was added to a target final concentration of 100 mM NaOH. The solution was mixed and incubated for 20 minutes to provide partial de-O-acetylation such that the O-acetylation level of the polysaccharide in the final conjugate was 0.8 to 1.4 μmol OAc / mg polysaccharide and / or a 50% to 60% reduction in the O-acetylation level relative to the starting material. Each monosaccharide repeat unit of the native MenC polysaccharide has two potential O-acetylation positions and generally has a total O-acetylation level of 40%-45% for all possible O-acetylation sites. A 50% reduction in O-acetyl groups relative to the starting material will result in a total O-acetylation level (all possible O-acetylation sites) of less than 25%.

[0440] The pH was adjusted to 6 and the temperature was lowered to 15°C. The dissolved polysaccharide was activated by adding sodium metaperiodate ( Figure 2B) so that the target concentration is 2mM. The pH is adjusted to 6 and the solution is mixed at 15°C. Periodate is oxidized and cleaved at the adjacent diol position to obtain a chain with an aldehyde at the end. The reaction is mixed until the average molecular size is reduced to 50,000 to 100,000 Daltons, as determined by HPSEC. The reducing activity (reflecting the amount of aldehyde) is 40 to 100nmol / mg polysaccharide. The reaction is quenched by adding glycerol in the amount of 0.5mL glycerol per gram of polysaccharide and mixing for a minimum of 5 minutes. First, a 5kDa MWCO regenerated cellulose filter column is used to concentrate the polysaccharide by ultrafiltration, and then diafiltration is performed for 8-12 volumes of 50mM sodium acetate buffer (pH 6.0) exchange. The material is further concentrated to a target concentration of 50mg / mL. The depolymerized / activated polysaccharide is filtered and stored.

[0441] The purified tetanus toxoid protein is concentrated to a target final concentration of up to 100 mg / mL through a 10 kDa MWCO PES membrane and then passed through a 0.2 micron filter. The filtered protein solution is stored at 1°C-5°C. The depolymerized / activated polysaccharide and concentrated tetanus protein are mixed together in a mass ratio of 0.5:1, 1:1, 2:1, 3:1, 4:1 or 5:1 (polysaccharide:protein). An aliquot of 100 mg / mL of sodium cyanoborohydride in 2.0 M phosphate buffer is added to the polysaccharide-protein mixture so that the sodium cyanoborohydride is 10 mg / mL and the phosphate buffer is 200 mM, pH 8.0. Saline is added to adjust the concentration, for example, the polysaccharide is adjusted to a target of 15-50 mg / mL. The reaction is mixed at 37°C ( Figure 2C ) for 16-30 hours. The reaction was diluted 1:2 with 6 mM phosphate buffered saline (PBS). An aliquot of 100 mg / mL sodium borohydride in 6 mM PBS was added to the reaction mixture to obtain a target of 0.5 mg sodium borohydride per mL reaction volume. The reaction was mixed at room temperature for a minimum of 15 minutes. Sodium borohydride caps the unreacted aldehyde by reducing it to an alcohol, resulting in a terminal unattached sugar with a primary hydroxyl group at position 7, or where the reducing end is modified with a (2-hydroxy)ethoxy group. The product (terminal sugar not shown) is shown in Figure 2D and Figure 2A The conjugate solution was diafiltered through a 50 kDa MWCO PES membrane against 10 volumes of 6 mM PBS exchange. The solution was stored at 1°C-5°C.

[0442] Ammonium sulfate was added to the conjugation reaction to obtain a 1M ammonium sulfate concentration. The pH was adjusted to 7 and mixed until dissolved at room temperature. The conjugated reaction mixture was applied to a HIC column filled with phenyl resin. Unconjugated polysaccharides were eluted with 2 to 7 column volumes of 1M ammonium sulfate solution. The conjugate was eluted with WFI. In this embodiment and subsequent embodiments, the HIC purification of the conjugate can provide a product in which less than 20% of the polysaccharides by mass are free (unconjugated) polysaccharides. The conjugate eluate was diafiltered against 10 volumes of 50mM sodium acetate (pH 6.0) exchange using a 100kDa MWCO PES membrane. The purified conjugate was finally filtered using a 0.2 micron membrane and stored at 1°C-5°C.

[0443] Example 2B

[0444] The purified capsular polysaccharide from Group C was dissolved in saline to a target concentration of 10 mg / mL. The solution was mixed until dissolved. The pH was adjusted to 6.0 and the temperature was changed to 15°C. The dissolved polysaccharide was activated by adding sodium metaperiodate ( Figure 2B ), so that the target concentration is 2mM. The reaction is mixed until the average molecular size is 50,000 to 100,000 Daltons, as determined by HPSEC. The reaction is quenched by adding glycerol in the amount of 0.5mL glycerol per gram of polysaccharide and mixing for a minimum of 5 minutes. First, a 5kDa MWCO regenerated cellulose filter column is used to concentrate the polysaccharide by ultrafiltration, and then diafiltration is performed for 8-12 volumes of 50mM sodium acetate buffer (pH 6.0) exchange. The material is further concentrated to a target concentration of 50mg / mL. The depolymerized / activated polysaccharide is filtered and stored at 1°C-5°C.

[0445] The purified tetanus toxoid protein is concentrated to a target final concentration of up to 100 mg / mL through a 10 kDa MWCO PES membrane and then passed through a 0.2 micron filter. The filtered protein solution is stored at 1°C-5°C. The depolymerized / activated polysaccharide and concentrated tetanus protein are mixed together in a molar ratio of 0.5:1, 1:1, 2:1, 3:1, 4:1 or 5:1 (polysaccharide:protein). An aliquot of 100 mg / mL of sodium cyanoborohydride in 2.0 M phosphate buffer is added to the polysaccharide-protein mixture so that the sodium cyanoborohydride is 10 mg / mL and the phosphate buffer is 200 mM, pH 8.0. Saline is added to adjust the concentration, for example, the polysaccharide is adjusted to a target of 15-50 mg / mL. The reaction is mixed at 37°C ( Figure 2C) for 16-30 hours. The reaction was diluted 1:2 with 6 mM phosphate buffered saline (PBS). An aliquot of 100 mg / mL sodium borohydride in 6 mM PBS was added to the reaction mixture to obtain a target of 0.5 mg sodium borohydride per mL reaction volume. The reaction was mixed at room temperature for a minimum of 15 minutes. Sodium borohydride caps the unreacted aldehyde by reducing it to an alcohol, resulting in a terminal unattached sugar with a primary hydroxyl group at position 7, or where the reducing end is modified with a (2-hydroxy)ethoxy group. The product (terminal sugar not shown) is shown in Figure 2D and Figure 2A The conjugate solution was diafiltered through a 50 kDa MWCO PES membrane against 10 volumes of 6 mM PBS exchange. The solution was stored at 1°C-5°C.

[0446] Ammonium sulfate was added to the conjugation reaction to obtain a 1M ammonium sulfate concentration. The pH was adjusted to 7 and mixed until dissolved at room temperature. The conjugated reaction mixture was applied to a HIC column filled with phenyl resin. Unconjugated polysaccharides were eluted with 2 to 7 column volumes of 1M ammonium sulfate solution. The conjugate was eluted with WFI. In this embodiment and subsequent embodiments, the HIC purification of the conjugate can provide a product in which less than 20% of the polysaccharides by mass are free (unconjugated) polysaccharides. The conjugate eluate was diafiltered against 10 volumes of 50mM sodium acetate (pH 6.0) exchange using a 100kDa MWCO PES membrane. The purified conjugate was finally filtered using a 0.2 micron membrane and stored at 1°C-5°C.

[0447] 3. Preparation of Group W-135 and Group Y Conjugates

[0448] Dissolve the purified capsular polysaccharide from group W-135 in sodium acetate buffer to a target concentration of 10 mg / mL. Mix the solution until dissolved. Heat the polysaccharide solution to 50°C-70°C using a jacketed heat exchanger. Adjust the pH to 4.5. Figure 4A , step 1) until the average molecular size is 150,000 Daltons, as determined by HPSEC. The reaction mixture is cooled to 1°C-5°C. Sodium metaperiodate is added to the polysaccharide solution to a target metaperiodate concentration of 2 mM ( Figure 4A , step 2). The pH is adjusted to 6.0 and the solution is mixed between 0 and 5°C for 60 minutes. Periodate oxidation and cleavage at adjacent diol positions yields an aldehyde, for example at position 7 of a sialic acid residue, such as Figure 4AAs shown. The reducing activity (reflecting the amount of aldehyde) is 60 to 150nmol / mg polysaccharide. The reaction is quenched by adding 0.5mL glycerol per gram of polysaccharide and mixing for a minimum of 5 minutes. The polysaccharide is concentrated by ultrafiltration using a 10kDa MWCO regenerated cellulose filter, and then diafiltration is performed for 10 volumes of 50mM sodium acetate buffer (pH 6.0) exchange. The material is further concentrated to a target concentration of 50mg / mL. The depolymerized / activated polysaccharide is filtered and stored at 1°C-5°C.

[0449] The purified tetanus toxoid protein is concentrated to a target final concentration of up to 100 mg / mL through a 10 kDa MWCO PES membrane and then passed through a 0.2 micron filter and stored at 1°C-5°C. The derivatized / activated polysaccharide and concentrated tetanus protein are mixed together in a mass ratio of 0.5:1, 1:1, 2:1, 3:1, 4:1 or 5:1 (polysaccharide:protein). An aliquot of 100 mg / mL sodium cyanoborohydride in 2.0 M phosphate buffer is added to the polysaccharide-protein mixture so that the sodium cyanoborohydride is 10 mg / mL and the phosphate buffer is 200 mM, pH 9.0. Saline is added to adjust the target concentration, for example, the polysaccharide is adjusted to a target of 15-50 mg / mL. The reaction ( Figure 4B ) and mixed overnight at room temperature.

[0450] The reaction was diluted 1:2 with 6 mM phosphate buffered saline (PBS). An aliquot of 100 mg / mL sodium borohydride in 6 mM PBS was added to the reaction mixture to achieve a target of 0.5 mg sodium borohydride per mL reaction volume. The reaction was mixed at room temperature for a minimum of 15 minutes. Sodium borohydride caps the unreacted aldehyde by reducing it to an alcohol. The product is shown in Figure 4C and Figure 3 middle.

[0451] Ammonium sulfate was added to the conjugation reaction to give a 1 M ammonium sulfate concentration. The pH was adjusted to 7 and mixed until dissolved at room temperature. The conjugation reaction mixture was applied to a HIC column filled with phenyl resin. Unconjugated polysaccharide was eluted with 2 to 7 column volumes of 1 M ammonium sulfate solution. The conjugate was eluted with WFI. The conjugate eluate was diafiltered using a 100 kDa MWCO PES membrane against 10 volumes of 50 mM sodium acetate (pH 6.0). The purified conjugate was finally filtered using a 0.2 micron membrane and the conjugate was stored at 1°C-5°C. The same process can be performed on the purified capsular polysaccharide of Group Y

[0452] 4. Preparation of quadrivalent vaccine

[0453] Example 4A

[0454] The tetravalent MenACYW-TT conjugate vaccine was formulated using the four monovalent PS-protein conjugates prepared as described in Examples 1A, 2A and 3-4 and diluted in sodium acetate buffered saline to a final concentration of 10 μg PS / serogroup / 0.5 mL. In other words, a 0.5 mL dose of the MenACYW conjugate vaccine contained 10 μg of each of meningococcal PS serogroups A, C, Y and W-135 conjugated to a total of 45 to 80 μg of tetanus toxoid protein (the actual amount of tetanus toxoid protein depends on the specific PS to protein ratio of each batch of monovalent mixed concentrate used in the formulation).

[0455] Each 0.5 mL dose of MenACYW conjugate vaccine was formulated in 30 mM sodium acetate-buffered saline solution (pH 6.0).

[0456] Example 4B

[0457] The tetravalent MenACYW-TT conjugate vaccine was formulated using the four monovalent PS-protein conjugates prepared as described in Examples 1A, 2B and 3-4 and diluted in sodium acetate buffered saline to a final concentration of 10 μg PS / serogroup / 0.5 mL. In other words, a 0.5 mL dose of the MenACYW conjugate vaccine contained 10 μg of each of meningococcal PS serogroups A, C, Y and W-135 conjugated to a total of 45 to 80 μg of tetanus toxoid protein (the actual amount of tetanus toxoid protein depends on the specific PS to protein ratio of each batch of monovalent mixed concentrate used in the formulation).

[0458] Each 0.5 mL dose of MenACYW conjugate vaccine was formulated in 30 mM sodium acetate-buffered saline solution (pH 6.0).

[0459] Example 4C

[0460] The tetravalent MenACYW-TT conjugate vaccine was formulated using the four monovalent PS-protein conjugates prepared as described in Examples 1B, 2B and 3-4 and diluted in sodium acetate buffered saline to a final concentration of 10 μg PS / serogroup / 0.5 mL. In other words, a 0.5 mL dose of the MenACYW conjugate vaccine contained 10 μg of each of meningococcal PS serogroups A, C, Y and W-135 conjugated to a total of 45 to 80 μg of tetanus toxoid protein (the actual amount of tetanus toxoid protein depends on the specific PS to protein ratio of each batch of monovalent mixed concentrate used in the formulation).

[0461] Each 0.5 mL dose of MenACYW conjugate vaccine was formulated in 30 mM sodium acetate-buffered saline solution (pH 6.0).

[0462] 5. Properties and Immunogenicity of Exemplary Conjugates

[0463] In the absence of an ADH linker, the MenA conjugate was prepared generally as described above, except that the polysaccharide concentration used in the conjugation reaction with tetanus toxoid was 12 mg / ml. The conjugate had a 0.3 polysaccharide / protein (PS / PR) mass ratio. The O-acetylation level was determined to be 3.0 μmol / mg polysaccharide.

[0464] In the case of an ADH linker, MenA conjugates were prepared generally as described above, except that the polysaccharide concentration used in the conjugation reaction with tetanus toxoid was 12 mg / ml. The conjugate had a 1.0 PS / PR mass ratio. The O-acetylation level was determined to be 2.8 μmol / mg polysaccharide. Several other batches of MenA conjugates with an ADH linker were prepared, in which the polysaccharide concentration used in the conjugation reaction with tetanus toxoid was 12 mg / ml or 16 mg / ml. The measured values ​​of the PS / PR mass ratio of these batches were 1.0, 1.1, 1.2 and 1.3, and the measured values ​​of the O-acetylation level in μmol / mg polysaccharide were 2.5, 2.8, 2.9 and 3.0. Some values ​​were observed more than once.

[0465] Each batch of MenA conjugates was demonstrated to be immunogenic (i.e., elicit anti-MenA antibodies in at least a substantial and statistically significant proportion of recipients as measured by serum bactericidal assay and / or ELISA) when administered in a tetravalent formulation to unvaccinated MenA human, mouse and / or guinea pig subjects relative to pre-treatment samples and / or non-immune controls.

[0466] MenC conjugates were generally prepared as described above. The conjugate had a polysaccharide / protein (PS / PR) mass ratio of 0.6. The O-acetylation level was determined to be 2.4 μmol / mg polysaccharide. Several other batches of MenC conjugates were prepared. The PS / PR mass ratios of these batches were measured to be 0.4, 0.6 and 0.7, and the O-acetylation levels in μmol / mg polysaccharide were measured to be 0.8, 1.2, 1.3, 1.4, 1.5, 2.2 and 2.3. Some values ​​were observed more than once.

[0467] Each batch of MenC conjugates was demonstrated to be immunogenic (i.e., elicit anti-MenC antibodies in at least a substantial and statistically significant proportion of recipients as measured by serum bactericidal assay and / or ELISA) when administered in a tetravalent formulation to unvaccinated human, mouse and / or guinea pig subjects relative to pre-treatment samples and / or non-immune controls.

[0468] MenW-135 conjugates were generally prepared as described above. The conjugate had a polysaccharide / protein (PS / PR) mass ratio of 0.9. The O-acetylation level was determined to be 1.6 μmol / mg polysaccharide. Several other batches of MenW-135 conjugates were prepared. The measured values ​​of the PS / PR mass ratios of these batches were 0.6, 0.7, 0.8, 0.9 and 1.2, and the measured values ​​of the O-acetylation levels in μmol / mg polysaccharide were 0.7, 0.8 and 1.3. Some values ​​were observed more than once.

[0469] Each batch of MenW-135 conjugates was demonstrated to be immunogenic (i.e., elicited anti-MenW-135 antibodies in at least a substantial and statistically significant proportion of recipients as measured by serum bactericidal assay and / or ELISA) when administered in a tetravalent formulation to unvaccinated human, mouse and / or guinea pig subjects relative to pre-treatment samples and / or non-immune controls.

[0470] MenY conjugates were generally prepared as described above. The conjugate had a polysaccharide / protein (PS / PR) mass ratio of 1.0. The O-acetylation level was determined to be 1.3 μmol / mg polysaccharide. Several other batches of MenY conjugates were prepared. The PS / PR mass ratios of these batches were measured to be 0.6, 0.7, 0.8 and 0.9, and the O-acetylation levels in μmol / mg polysaccharide were measured to be 0.8, 0.9, 1.0, 1.1 and 1.3. Some values ​​were observed more than once.

[0471] Each batch of MenY conjugates was demonstrated to be immunogenic (i.e., elicited anti-MenY antibodies in at least a substantial and statistically significant proportion of recipients as measured by serum bactericidal assay and / or ELISA) when administered in a tetravalent formulation to unvaccinated human, mouse and / or guinea pig subjects relative to pre-treatment samples and / or non-immune controls.

[0472] All the conjugate batches described above are populations of conjugate molecules with a weight average molecular weight ranging from 300 to 1500 kDa.

[0473] 6. Clinical trials

[0474] The tetravalent MenACYW-TT conjugate as described herein was used in clinical studies to evaluate the safety and immunogenicity of different vaccination schedules in infants (6 weeks of age and older) and adults aged 56 years and older.

[0475] a) Phase I / II Clinical Trial 1 - Safety and Immunogenicity of Various MenACYW-TT-Like Formulations Administered to Young Children Unvaccinated Against Meningococcal Vaccine (12 Months + / - 21 Days)

[0476] This Phase I / II study evaluated the safety and immunogenicity of a single dose of each tetravalent meningococcal polysaccharide-tetanus toxoid conjugate formulation associated with MenACYW-TT administered intramuscularly to infants 12 months + / - 21 days of age (Groups 1-5). (fully de-O-acetylated meningococcal group C polysaccharide-tetanus toxoid conjugate, referred to herein as "MenC-TT", a licensed monovalent meningococcal conjugate vaccine) was administered to the control group (Group 6).

[0477] Formulation 1A contained 4 μg of polysaccharide of each of the four polysaccharides (i.e. MenACYW) and 22.1 μg of TT per 0.5 mL dose. All had native O-acetylation levels and were conjugated essentially as described above by periodate activation and reductive amination (MenCYW) or using carbonyldiimidazole and adipic acid dihydrazide (MenA).

[0478] Formulation 1B contained 10 μg each of MenA and MenW polysaccharides and 4 μg each of MenC and MenY polysaccharides and 36.6 μg TT per 0.5 mL dose. Otherwise identical to formulation 1A.

[0479] Formulation 1C contained 10 μg of each of the four polysaccharides and 54.8 μg of TT per 0.5 mL dose. Other aspects were the same as Formulation 1A.

[0480] Formulation 2A contained 4 μg of polysaccharide (of each of the four polysaccharides) and 33.9 μg of TT per 0.5 mL dose. MenC, Y and W polysaccharides were partially de-O-acetylated by alkaline treatment and conjugated by periodate activation and reductive amination essentially as described above. MenA polysaccharide has a native O-acetylation level and was prepared as a neoglycoconjugate using conjugation chemistry essentially as described in Example 5 of US2005 / 0002957.

[0481] Formulation 2B contained 10 μg of each of the four polysaccharides and 84.8 μg of TT per 0.5 mL dose. Other aspects were the same as Formulation 2A.

[0482] All of the above formulations contained 0.67% NaCl and were buffered with pH 6 sodium phosphate.

[0483] The six study groups are summarized and characterized in Table 1 .

[0484]

[0485] The male to female ratio varied among treatment groups, with extremes ranging from 39.3% to 60.7% in Group 2 and from 62.3% to 37.7% in Group 5. The age range for all groups was 11.0 to 12.0 months; the mean for each group was 11.5 to 11.7 months.

[0486] Safety data were collected as unsolicited adverse events (AEs) 30-37 days after vaccine administration. The interval for solicited AEs was between D0 and D7 (i.e., 0 to 7 days after administration). The collection of solicited reactogenicity included injection site tenderness, redness, and swelling, as well as fever, vomiting, loss of appetite, abnormal crying, lethargy, and irritability.

[0487] All vaccinated subjects completed the study, but 56 vaccinated subjects (all groups) were excluded from the analysis due to protocol deviations, the most common reason being failure to provide a blood sample on Days 30-37 within the specified time window.

[0488] Two subjects experienced immediate unsolicited AEs, one of which was considered related to the vaccination (rash in one subject in Group 1).

[0489] Solicited Injection Site Reactions Between Day 0 and Day 7: The majority of subjects in all groups experienced solicited reactions. Overall, the rates of solicited reactions were comparable between groups. The percentage of recipients in Groups 1-5 who reported an injection site reaction was similar to that seen for subjects who received the control vaccine (Group 6).

[0490] There was no clear correlation between the injection site reaction rate and the amount of tetanus toxoid contained in each vaccine formulation.

[0491] The most common injection site reaction was tenderness, with rates ranging from 25.4% in Group 1 to 39.3% in Group 5; the next was erythema, ranging from 29.5% in Group 2 to 39.3% in Group 5; and the least common reaction was swelling, ranging from 13.6% in Group 4 to 23.0% in Group 3. Most solicited injection site reactions were Grade 1 in intensity, began and improved within 3 days of vaccination, and did not require any intervention. No group trends were seen in terms of intensity, time of onset, duration, or actions taken.

[0492] Solicited Systemic Reactions Between Days 0 and 7: As with injection site reactions, the overall incidence of systemic reactions such as fever, vomiting, abnormal crying, lethargy, loss of appetite, and irritability were comparable between Groups 1-5 and similar to those seen with the control vaccine. The most common systemic reaction in all groups was irritability, ranging from 54.2% in Group 4 to 70.5% in Groups 2 and 5. The next most common (all except Group 2) was abnormal crying, ranging from 34.4% in Group 2 to 48.4% in Group 6, followed by loss of appetite (which was the second most common reaction in Group 2), ranging from 27.9% in Group 6 to 46.8% in Group 6. Vomiting rates ranged from 18.6% in Group 4 to 32.3% in Group 6; and fever was the least reported reaction, ranging from 11.1% in Group 1 to 25.8% in Group 6. Most solicited systemic reactions were Grade 1 in intensity, began and improved within 3 days of vaccination, and did not require any intervention. No group trends were seen with respect to intensity, onset, duration, or actions taken.

[0493] Unsolicited Adverse Events Between Day 0 and Day 30: A total of 931 unsolicited AEs were reported by 327 subjects. A total of 107 unsolicited AEs were considered related to vaccination and identified as adverse reactions (ARs). The most common ARs in the SOC were 1) systemic disorders and administration site reactions (25 cases); 2) infections and infestations and skin and subcutaneous tissue disorders (20 cases each); and 3) gastrointestinal disorders and respiratory, thoracic and mediastinal disorders (16 cases each). There was no clear correlation between the number of ARs and the vaccine received. A total of 4 systemic ARs of grade 3 intensity were reported: 1 case each in Group 1 and Group 4, and 2 cases in Group 5.

[0494] Serious AEs: No deaths occurred. During the study, a total of 7 SAEs occurred in 7 subjects: 2 in subjects in Group 1, 1 in subjects in Group 2, 2 in subjects in Group 3, and 2 in subjects in Group 4. No SAEs were reported in Groups 5 and 6. One of these SAEs, reactive arthritis experienced by a subject in Group 3, was considered related to the vaccination. The subject fully recovered after 23 days.

[0495] The conclusion regarding safety was that the single dose was well tolerated in all groups with no significant differences in the safety profile.

[0496] Functional antibodies to meningococcal serogroups A, C, Y, and W-135 were measured by serum bactericidal assays using human complement (SBA-HC) and baby rabbit complement (SBA-BR) to determine 1) the proportion of subjects with SBA-HC titers ≥1:8 and ≥1:4, or SBA-BR titers ≥1:8; 2) the geometric mean titer (GMT); 3) the reverse cumulative distribution curve (RCDC); and 4) the distribution of titers. In addition, antibody titers to tetanus toxoid present in the vaccine were assessed by ELISA. Blood samples collected before vaccination ("before" in Tables 2 and 3) and on days 30-37 after vaccination ("after" in Tables 2 and 3) were assayed.

[0497] The SBA-HC results are shown in Tables 2 and 3. In Tables 2 and 3, the 95% confidence intervals are percentages.

[0498]

[0499]

[0500]

[0501] As can be seen in the table, pre-vaccination titers were low for all serogroups. For serogroup A, most subjects in all groups had titers of 8 or less; for the other three serogroups, almost all subjects had values ​​<4. Post-vaccination in Groups 1-5, most titer values ​​were between 8 and 128 for serogroup A; between 16 and 1024 for serogroup C; between <4 and 128 for serogroup Y; and between <4 and 64 for serogroup W-135. For the control group, values ​​for serogroups A, Y, and W-135 were mostly between <4 and 4, while for serogroup C, most values ​​were between 256 and 1024. The high-dose groups tended to have higher antibody responses compared to the low-dose groups.

[0502] In the SBA-BR assay, GMTs were comparable across treatment groups prior to dosing, ranging from 4.22 to 5.26 for serogroup A, 4.00 to 6.26 for serogroup C, and 6.01 to 8.45 for serogroup W-135. Higher variability was seen in serogroup Y, with values ​​ranging from 17.36 (group 5) to 35.92 (group 1).

[0503] In groups 1-5, the values ​​after serogroup A vaccination ranged from 336.91 (group 1) to 759.35 (group 5); the value in the control group was 5.66. The values ​​for serogroup C ranged from 145.53 (group 1) to 636.37 (group 5). The value in the control group was 1290.16. The values ​​for serogroup Y ranged from 586.54 (group 3) to 713.70 (group 4). The value in the control group was 23.41. The values ​​for serogroup W-135 ranged from 912.28 (group 4) to 1518.71 (group 5). The value in the control group was 8.57.

[0504] b) Phase II clinical trial 1-Safety and immunogenicity of MenACYW-TT in infants and young children

[0505] A Phase II randomized, open-label, multicenter clinical trial was conducted in 580 children in the United States. The trial was designed to investigate the safety and immunogenicity profile of MenACYW-TT vaccine when given at different schedules and concurrently with routine pediatric vaccinations. The study also aimed to describe the safety and immunogenicity profile of MenACYW-TT vaccine and selected licensed pediatric vaccines ( (DTaP-IPV / Hib), (PCV7) or 13 (PCV13), II(MMR) (V) or RECOMBIVAX (HepB), (RV1) and Immunogenicity profile of (RV5).

[0506] Participants received MenACYW-TT vaccine concurrently with routine vaccines (study group; groups 1-5 in Table 1) or routine pediatric vaccines alone (control group; groups 6-7).

[0507] Infants were randomly assigned to 3 study groups (Groups 1-3) and 2 control groups (Groups 6 and 7) at 2 months of age as shown in Table 1. Infants in study groups 1-3 received 3 or 4 doses of MenACYW-TT vaccine (concurrently with routine vaccines) as described in Table 4.

[0508] Table 4 provides a summary of the clinical trial design.

[0509]

[0510] Six-month-old infants who received two doses of MenACYW-TT vaccine at 6 and 12 months of age (simultaneous with routine vaccines) (Group 4) and 12-month-old infants who received one dose of MenACYW-TT vaccine at 12 months of age (simultaneous with routine vaccines) (Group 5) were also recruited.

[0511] Serum bactericidal assays with human (hSBA) and rabbit (rSBA) complement were used to measure antibodies against meningococcal serogroups A, C, Y and W at baseline and 30 days after the last infant dose. The lower limit of quantitation (LLOQ) for both assays was 1:4. See, e.g., Maslanka et al., Standardization and a Multilaboratory Comparison of Neisseria meningitidis Serogroup A and C Serum Bactericidal Assays, Clinical and Diagnostic Laboratory Immunology, March 1997, pp. 156-167 and Goldschneider Gotschlich and Artenstein, Immunity to Meningococcus, The Role of Human Antibodies, the Journal of Experimental Medicine, 1969.

[0512] Safety data were collected for up to 6 months after the last dose of vaccine. The interval for solicited adverse events (AEs) was between D0 and D7 (i.e., 0 to 7 days after dosing). The collection of solicited reactogenicity included daily measurement of body temperature and injection site redness and swelling, as well as recording the intensity of injection site pain, headache, myalgia, and discomfort. Unsolicited AEs and serious adverse events (SAEs) were also collected throughout the study. All statistical analyses were descriptive.

[0513] Demographic characteristics are analyzed for the safety analysis set, which is defined as the subject who receives at least one dose of study vaccine or control vaccine and can obtain safety data. The mean age of inclusion in the study of Group 1, Group 2, Group 3, Group 6 and Group 7 was 2.19 months, 2.20 months, 2.24 months, 2.18 months and 2.20 months, respectively. The age range of Group 1 was 1.57 months to 2.97 months, Group 2 was 1.57 months to 2.90 months, Group 3 was 1.53 months to 3.00 months, Group 6 was 1.53 months to 2.87 months, and Group 7 was 1.70 months to 2.97 months (inclusion criteria 2 months [42 days to 89 days]). In Group 4, the mean age was 6.23 months, and the age range was 5.63 months to 6.50 months (inclusion criteria 6 months [180 days ± 14 days]). In Group 5, the mean age was 12.4 months, and the age range was 12.2 months to 12.7 months (inclusion criterion 12 months [365 days + 14 days]).

[0514] After completing the infant series and receiving additional MenACYW-TT vaccine doses in the second year of life (Groups 1-4), the majority of study participants achieved 1:8 protective titers (91%-100% for human complement [hSBA] and 80%-100% for baby rabbit complement [rSBA]) for all four serogroups (ACYW) included in the MenACYW-TT vaccine, regardless of the number of doses received in the first year of life. For participants who received a single dose at 12 months of age (Group 5), 1:8 ACYW protective titers ranged from 47.5%-90% (hSBA) to 62%-100% (rSBA). Thus, the MenACYW-TT conjugate vaccine demonstrated a robust immunogenic response following additional dosing in the second year of life, regardless of the initial schedule received in the first year of life.

[0515] Figure 6 The percentage of subjects in Groups 1-4 with hSBA levels greater than or equal to 1:8 for each of serotypes A, C, Y, and W is provided. Figure 7 Similar rSBA results for these same groups are provided.

[0516] Following a single dose of MenACYW-TT vaccine at 12 months of age (Group 5), the immune response to hSBA was similar to that seen in the 3-dose series (Group 3) for serogroup C (90%), but was lower for serogroups Y (47.5%), A (75%) and W-135 (54%). See Table 5, which shows the hSBA and rSBA titers for subjects who received a single dose of MenACYW-TT vaccine at 12 months of age (Group 5).

[0517]

[0518] There was no evidence that co-administration of MenACYW-TT vaccine interfered with routine paediatric vaccines (data not shown).

[0519] The frequency of solicited injection site reactions did not increase with repeated doses of the vaccine. Figure 8 , which presents the cumulative percentage of participants reporting one or more solicited injection site reactions within 7 days of administration of the MenACYW-TT vaccine. The cumulative percentage of participants reporting ≥1 solicited injection site reaction within 7 days of administration of the MenACYW-TT vaccine was highest in the groups that received 4 doses (Groups 1 and 2, 80.0%-80.8%), followed by the groups that received 2 doses (Group 4, 75.3%) or the groups that received 3 doses (Group 2, 74.0%), and was lowest in the group that received 1 dose (Group 5, 57.4%).

[0520] Fig. 9 Solicited systemic reactions within 7 days of administration of MenACYW-TT vaccine plus conventional vaccine or conventional vaccine alone are shown.

[0521] Most non-serious adverse events (NSAEs) were reported following vaccination with MenACYW-TT or conventional vaccines, and every Grade 3 NSAE reported was not related to the study vaccine. No serious adverse events were reported to be related to the vaccine.

[0522] When MenACYW-TT and conventional vaccines were given simultaneously (ie on the same day, as separate vaccines), both were immunogenic compared to a control group given conventional vaccine without MenACYW-TT vaccine, indicating that there is no negative interaction between MenACYW-TT and conventional vaccines.

[0523] In conclusion, MenACYW-TT vaccine was safe and well tolerated in infants and young children, regardless of the immunisation schedule and number of doses given. The safety profile of the MenACYW-TT vaccine was generally similar to that of the control group, regardless of the immunisation schedule and number of doses given.

[0524] Conclusions: The investigational MenACYW-TT vaccine was well tolerated and immunogenic. All vaccination schedules, including one or more doses in the first and second year of life, induced robust immune responses against all four N. meningitidis serogroups A, C, Y and W, with an acceptable safety profile.

[0525] c) Phase II Clinical Trial 2 - Safety and Immunogenicity of MenACYW-TT in Adults 56 Years and Older

[0526] Age and underlying chronic illness are important risk factors for meningococcal disease, so the risk is increased in older adults. A clinical study was conducted in adults aged 56 years and older to evaluate the efficacy of a single-dose meningococcal vaccine (MPSV4) compared with the licensed quadrivalent meningococcal common polysaccharide vaccine (MPSV4). - Safety and immunogenicity of the MenACYW-TT vaccine compared with A / C / Y / W-135.

[0527] A randomized, open-label, multicenter, phase II study was conducted in 301 healthy adults aged 56 years or older in the United States. Participants at 12 centers were randomly assigned to receive either one dose of MenACYW-TT or MPSV4 ( -A / C / Y / W-135). Patients were divided into 2 subsets based on age: 1) 56 to 64 years and 2) greater than or equal to 65 years.

[0528] Four study groups were formed as follows. Group 1a (n=101, age 56-64 years) and Group 1b (n=100, ≥65 years) received MenACYW-TT. Group 2a (n=50, age 56-64 years) and Group 2b (n=50, ≥65 years) received MPSV4 vaccine. "Group 1" will refer to Group 1a and Group 1b. "Group 2" will refer to Group 2a and Group 2b. The above demographic results are summarized in Table 6.

[0529]

[0530] As described above, serum bactericidal assays (SBA) performed with human complement (hSBA) and baby rabbit complement (rSBA) were used to measure antibodies against meningococcal serogroups A, C, Y, and W at baseline and 30 days after vaccine administration. The lower limit of quantitation (LLOQ) for both assays was 1:4.

[0531] Safety data for up to 30 days were collected after administration. The interval for solicited AEs was between D0 and D7 (i.e., 0 to 7 days after administration). The collection of solicited reactogenicity included daily measurement of body temperature and injection site redness and swelling, as well as recording the intensity of injection site pain, headache, myalgia, and discomfort. Unsolicited AEs and serious adverse events (SAEs) were also collected throughout the study. All statistical analyses were descriptive.

[0532] Demographic characteristics were analyzed for the safety analysis set, which was defined as subjects who received at least one dose of the study vaccine or control vaccine and for whom safety data were available. At the time of recruitment, the mean age of subjects in Groups 1 and 2 was similar (66.1 ± 7.13 years and 65.8 ± 6.58 years, respectively). In addition, the age of the group of subjects 56-64 years old and the group of subjects ≥ 65 years old was similar (60.3 ± 2.52 years in Group 1a, 60.8 ± 2.59 years in Group 2a, 71.9 ± 5.28 years in Group 1b, and 70.8 ± 5.45 years in Group 2b).

[0533] There were slightly more female subjects (60.8% [121 / 199] and 55.0% [55 / 100], respectively) than male subjects (39.2% [78 / 199] and 45.0% [45 / 100]) in both groups 1 and 2. The same trend was observed in the subsets, except for group 2b, in which the number of female and male subjects was equal (50.0% [25 / 50]).

[0534] Across all subgroups, the percentage of study participants with hSBA titers ≥1:8 for serogroups A, C, Y, and W-135 increased significantly at Day 30 compared with baseline. Results within each vaccination group were generally similar in both age strata (56 to 64 years and ≥65 years), as shown in Table 7.

[0535]

[0536] Fig.10 The percentage of subjects achieving hSBA levels ≥ 1:8 at D30 for serogroups A, C, Y and W is presented (using data from all patients (ie age groups combined)). For serogroups A and C, the percentage of individuals with hSBA titers ≥ 1:8 following MenACYW-TT was comparable to the titers following MPSV4. For serogroups Y and W, the percentage of individuals with hSBA titers ≥ 1:8 following MenACYW-TT was higher than the titers following MPSV4.

[0537] Fig.11 The geometric mean titers (GMTs) of the two vaccines against the different serogroups at D30 are provided. For all serogroups, the GMTs of MenACYW-TT were greater than or equal to the GMTs of MPSV4.

[0538] For all four vaccine groups, the percentage of participants with an rSBA titer greater than or equal to 1:8 was comparable between MenACYW-TT recipients and MPSV4 recipients. Fig.12 .

[0539] Solicited injection site reactions ( Fig.13 ) and solicited systemic reactions ( Fig.14 ) is similar to administering the MPSV4 vaccine.

[0540] Overall, the reactogenicity profiles of the MenACYW-TT and MPSV4 vaccines were similar. Most unsolicited adverse events were grade 1 or 2 in intensity. There were no immediate hypersensitivity reactions and discontinuations attributed to AEs or SAEs. No increase in reactogenicity was observed in elderly vaccine recipients. No serious adverse events were reported.

[0541] MenACYW-TT was well tolerated and immunogenic when given to adults aged 56 years or older. Therefore, MenACYW-TT represents an alternative vaccine for the prevention of invasive meningococcal disease, including in areas of the world where only common polysaccharide vaccines (such as MPSV4) are currently available for immunization of older adults.

[0542] d) Phase II clinical trial 3 - Safety and immunogenicity of MenACYW-TT administered to healthy, unvaccinated young children (12-23 months)

[0543] The MenACYW-TT conjugate vaccine is intended for use in individuals 6 weeks of age and older. The safety and immunogenicity of a single dose in young children was evaluated using a licensed quadrivalent meningococcal conjugate vaccine (MCV4-TT) as a control.

[0544] A phase II, randomized, open-label study was conducted in Finland in 188 young children (12-23 months of age) who had not received meningococcal vaccination. Participants were randomized to receive one dose of MenACYW-TT vaccine or MCV4-TT. Antibodies to meningococcal serogroups A, C, W, and Y were measured at baseline and 30 days after dosing using serum bactericidal assays with human (hSBA) and baby rabbit (rSBA) complement. The LLOQ for both bactericidal assays was 1:4. Antibody responses to tetanus were also measured.

[0545] Safety data were collected for up to 30 days after dosing. The interval for solicited adverse events (AEs) was between D0 and D7. The collection of solicited reactogenicity included daily measurement of body temperature and injection site redness and swelling, as well as recording the intensity of injection site pain, headache, myalgia, and discomfort. Unsolicited AEs and serious adverse events (SAEs) were collected throughout the study. All analyses were descriptive.

[0546] Table 8 provides data on the study design and subject disposition of this trial.

[0547]

[0548]

[0549] Demographic analyses were performed on the safety analysis set. The safety analysis set was defined as subjects who received at least one dose of study vaccine or control vaccine and for whom safety data were available. There were 98 (52.1%) male subjects and 90 (47.9%) female subjects in the safety analysis set; the overall ratio of male / female subjects was 1.09. There were more males than females in the MenACYW-TT group (male / female ratio, 1.54). There were more females than males in the MCV4-TT group (male / female ratio, 0.77). The age of the subjects was comparable between the two groups. At recruitment, the mean age of the subjects was 1.44±0.302 years in the MenACYW-TT group and 1.47±0.314 years in the MCV4-TT group.

[0550] For serogroups A, W, and Y, the percentage of subjects with hSBA vaccine serological responses to MenACYW-TT vaccine was comparable to that for MCV4-TT [range 96.7% to 98.9% (MenACYW-TT) and 91.9% to 98.8% (MCV4-TT)] ( Fig.15 The serum response to hSBA vaccine was defined as follows: a baseline titer of <1:8 and a post-vaccination titer of ≥1:8 or a baseline titer of ≥1:8 and a ≥4-fold increase after vaccination.

[0551] For serogroup C, the percentage of subjects with seroresponses to MenACYW-TT (100.0%) was higher than that for MCV4-TT (86.0%). Using rSBA, the trend for serogroup C was similar.

[0552] Data on the percentage of subjects achieving hSBA titers >=8 (>=1:8) at D30 post-vaccine are presented in Table 9.

[0553]

[0554] MenACYW-TT elicited comparable immune responses to serogroups A, W, and Y, and a higher immune response to serogroup C, when evaluated using hSBA geometric mean titers and the percentage of subjects with an hSBA titer ≥ 8 (>=1:8) after vaccination ( Fig.16 and Table 10). Fig.16 and Table 10 show the same data, except that the data in Table 10 are converted to the log2 scale, as shown in Fig.16 shown.

[0555]

[0556] Safety was also evaluated in this study. The reactogenicity profile was comparable between the two vaccines. The percentage of subjects reporting at least 1 solicited injection site reaction was comparable between the two vaccines (48.9% and 53.2%). Data related to injection site erythema, tenderness, and swelling were shown in Fig.17 Moderate. Most reactions at the injection site were grade 1 or 2 in intensity, all started between D0 and D03, and most lasted 1 to 3 days.

[0557] Few subjects reported Grade 3 solicited injection site reactions: 3.2% of subjects in the MenACYW-TT group and 4.3% of subjects in the MCV4-TT group.

[0558] Solicited systemic reactions were also similar between the two groups ( Fig.18 ).

[0559] The percentage of subjects reporting at least 1 unsolicited non-serious AE was comparable between study groups. Most unsolicited adverse events were grade 1 or 2 in intensity. No immediate unsolicited AEs were reported in either group. There were no immediate SAEs, including any hypersensitivity events or life-threatening events. The two serious adverse events reported were considered not related.

[0560] Conclusions: The MenACYW-TT vaccine was well tolerated and immunogenic. The single-dose MenACYW-TT vaccine demonstrated excellent potential as an alternative vaccine option for young children receiving meningococcal vaccine for the first time.

[0561] e) Phase II clinical trial 4 - Safety and immunogenicity of MenACYW-TT administered to healthy, unvaccinated adolescents (10-18 years)

[0562] This Phase II study evaluated the safety and immunogenicity of a single dose (10 μg polysaccharide per serogroup, conjugated to a total of 65 μg TT, buffered to pH 6.0 in 0.67% NaCl / 30 mM sodium acetate) of MenACYW-TT administered intramuscularly to adolescents aged 10-18 years. (Meningococcal (groups A, C, Y and W-135) oligosaccharide diphtheria CRM197 conjugate vaccine, referred to herein as "MenACYW-CRM 197 ”, a licensed quadrivalent meningococcal conjugate vaccine) was given to the control group (Group 2). MenACYW-TT will also be given in combination with and (Group 3) had the same effect as that of single administration and The control vaccine and and Vaccines. Table 11 summarizes and characterizes the four study groups.

[0563]

[0564] *: First dose of HPV vaccine was given on D0; HPV dose 2 and dose 3 were given 2 months and 6 months after dose 1, respectively.

[0565] A total of 74 subjects (4.3%) did not complete the trial: 10 (2.0%) in Group 1, 7 (1.4%) in Group 2, 27 (6.7%) in Group 3, and 30 (10.0%) in Group 4. The most commonly reported reasons for discontinuation were: voluntary discontinuation not due to adverse events, loss to follow-up, and noncompliance with the protocol. There were no early discontinuations due to SAEs or other AEs.

[0566] Antibodies to meningococcal serogroups A, C, W, and Y at baseline and 30 days post-dose were measured using a serum bactericidal assay with human (hSBA) complement. The LLOQ for the bactericidal assay was 1:4. hSBA data were collected for 463 members of Group 1, 464 members of Group 2, and 360 members of Group 3. The hSBA results are in Table 12, where the % subjects indicate the percentage of subjects with a positive serological response, i.e., subjects with a pre-vaccination hSBA titer <1:8 who had a post-vaccination hSBA ≥1:8, or subjects with a pre-vaccination titer ≥1:8 who had at least a 4-fold increase in hSBA titer from pre-vaccination to post-vaccination. For all four, the percentage of subjects showing a positive serological response to MenACYW-TT was higher than for serogroups MenACYW-CRM 197 situation.

[0567]

[0568] The difference in serological response frequency between Group 1 and Group 2 along with its 95% confidence interval is shown in

[0569] Table 13.

[0570]

[0571] The difference in serological response frequency between groups 1 and 3 was not significant at the 95% confidence level, which is consistent with the effectiveness of MenACYW-TT being unaffected by and The conclusions on the effects of combined administration were consistent.

[0572] Table 14 shows the hSBA results expressed as geometric mean titers (GMTs) at day 0 (D0) and day 30 (D30), along with 95% confidence intervals.

[0573]

[0574] All groups were compared for immune responses to diphtheria and tetanus. The results are shown in Table 15, expressed as geometric mean concentration (GMC); % subjects with > 0.1 IU / mL; and % subjects with anti-tetanus antibody and anti-diphtheria antibody concentrations > 1.0 IU / mL.

[0575]

[0576]

[0577] The results were similar to those in group 3, where MenACYW-TT was given in combination with The conclusion is consistent with that of not interfering with the immunogenicity of the latter (see the results of Group 4).

[0578] Vaccine responses have also been characterized for the following antigens: pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertussis neferin (PRN), and pertussis fimbrial antigen (FIM).

[0579] Table 16.

[0580]

[0581] The following items were observed in terms of safety: the occurrence, nature, duration, intensity and relationship to vaccination of any unsolicited systemic adverse events (AEs) reported within 30 minutes after vaccination; the occurrence, onset time, day of occurrence, intensity, actions taken and whether the reaction led to early termination of the study for solicited injection site reactions occurring up to 7 days after vaccination on D0; the occurrence, onset time, day of occurrence, intensity, actions taken and whether the reaction led to early termination of the study for solicited systemic reactions occurring up to 7 days after vaccination on D0; the occurrence, nature, onset time, duration, intensity, actions taken, relationship to vaccination (only for systemic AEs) of unsolicited AEs up to 23-37 days after vaccination on D0, and whether the event led to early termination of the study; the occurrence, nature, onset time, duration, severity criteria, relationship to vaccination, outcomes and whether serious adverse events (SAEs) led to early termination of the study during the entire trial from D0 vaccination to up to 180 days (Groups 1 and 2) or 210 days (Groups 3 and 4) after vaccination on D0. Solicited systemic reactions included fever, myalgia, and headache. Solicited injection site reactions included pain, erythema, and swelling.

[0582] The percentage of subjects reporting at least 1 solicited reaction between D0 and D07 was significantly higher in the MenACYW-TT conjugate vaccine and The percentages of subjects reporting at least 1 solicited reaction were comparable between Group 1 (63.5%) (315 / 496) and Group 2 (64.2%) (316 / 492), respectively. The percentages of subjects reporting at least 1 solicited reaction were comparable between subjects receiving the MendACYW-TT conjugate vaccine concurrently with Tdap and HPV and those receiving Tdap and HPV alone: ​​88.9% (345 / 388) and 89.0% (258 / 290), respectively, in Group 3 and Group 4. The percentages of subjects reporting at least 1 solicited injection site reaction were comparable between Groups 1, 2, and 3: 46.6% (231 / 496), 45.7% (225 / 492), and 49.0% (190 / 388), respectively. No increase in local reactivity was seen with the MenACYW-TT conjugate vaccine when it was given simultaneously with Tdap and HPV (Group 3) compared to when it was given alone (Group 1).

[0583] The most commonly reported solicited injection site reaction was pain, reported by 45.2% (224 / 496) of subjects in Group 1, 42.5% (209 / 492) of subjects in Group 2, and 47.2% (183 / 388) of subjects in Group 3, followed by injection site erythema, reported by 5.0% (25 / 496) of subjects in Group 1, 7.5% (37 / 491) of subjects in Group 2, and 3.9% (15 / 388) of subjects in Group 3, and injection site swelling, reported by 5.4% (27 / 496) of subjects in Group 1, 6.5% (32 / 491) of subjects in Group 2, and 4.4% (17 / 388) of subjects in Group 3. Most injection site reactions were of grade 1 or 2 intensity, began between D0 and D03, and lasted 1 to 3 days. The percentage of subjects with any Grade 3 injection site reaction was 1.8% (9 / 496) in Group 1, 2.2% (11 / 492) in Group 2, and 2.8% (11 / 388) in Group 3. The percentage of subjects with Grade 3 pain at the injection site was 1.4% (7 / 496) in Group 1, 1.0% (5 / 492) in Group 2, and 2.3% (9 / 388) in Group 3. The percentage of subjects with Grade 3 erythema was 0.4% (2 / 496) in Group 1, 1.2% (6 / 491) in Group 2, and 0.5% (2 / 388) in Group 3. The percentage of subjects with Grade 3 swelling was 0.2% (1 / 496) in Group 1, 0.4% (2 / 491) in Group 2, and 0.3% (1 / 388) in Group 3. The intensity grades generally have the following meanings. Grade 1: Does not interfere with activities. Grade 2: Somewhat interferes with activities. Grade 3: Severely interferes with daily activities.

[0584] The percentage of subjects reporting at least 1 solicited systemic reaction following vaccination was comparable between Group 1 (52.0% [258 / 496]) and Group 2 (51.0% [251 / 492]). Myalgia was the most commonly reported solicited systemic reaction, followed by headache and malaise, with fever reported less frequently. Myalgia was reported by 35.3% (175 / 496) of subjects in Group 1 and 35.2% (173 / 492) of subjects in Group 2. Headache was reported by 30.2% (150 / 496) of subjects in Group 1 and 30.9% (152 / 492) of subjects in Group 2. Malaise was reported by 26.0% (129 / 496) of subjects in Group 1 and 26.4% (130 / 492) of subjects in Group 2. Fever was reported by 1.4% (7 / 494) of subjects in Group 1 and 1.2% (6 / 488) of subjects in Group 2.

[0585] The percentage of subjects who experienced at least 1 solicited systemic reaction after vaccination was comparable between Group 3 (70.6% [274 / 388]) and Group 4 (65.9% [191 / 290]). Myalgia was the most commonly reported systemic reaction: 61.3% (238 / 388) of subjects in Group 3 and 55.4% (160 / 289) of subjects in Group 4. Headache was reported by 33.8% (131 / 388) of subjects in Group 3 and 29.0% (84 / 290) of subjects in Group 4. Malaise was reported by 29.1% (113 / 388) of subjects in Group 3 and 27.9% (81 / 290) of subjects in Group 4. Fever was reported by 1.6% (6 / 387) of subjects in Group 3 and 0.7% (2 / 286) of subjects in Group 4. Overall, most solicited systemic reactions were grade 1 or 2 in intensity, began between D0 and D03, and lasted 1 to 3 days.

[0586] Overall, the percentage of subjects reporting Grade 3 solicited systemic reactions was comparable between Group 1 (3.8% [19 / 496]) and Group 2 (4.3% [21 / 492]). The percentage of subjects reporting Grade 3 solicited systemic reactions was comparable between Group 3 (7.5% [29 / 388]) and Group 4 (5.5% [16 / 290]). The most commonly reported Grade 3 systemic reaction was myalgia, followed by malaise and headache. The percentage of subjects reporting Grade 3 myalgia was comparable between Group 1 (1.6% [8 / 496]) and Group 2 (1.8% [9 / 492]) and between Group 3 (4.6% [18 / 388]) and Group 4 (3.8% [11 / 289]). The percentage of subjects reporting Grade 3 discomfort was comparable between Group 1 (2.2% [11 / 496]) and Group 2 (2.8% [14 / 492]). Discomfort was reported more frequently in Group 3 (2.6% [10 / 388]) than in Group 4 (1.7% [5 / 290]). The percentage of subjects reporting Grade 3 headache was comparable in Group 1 (1.8% [9 / 496]) and Group 2 (1.8% [9 / 492]). Headache was reported more frequently in Group 3 (2.8% [11 / 388]) than in Group 4 (1.7% [5 / 290]).

[0587] Overall, the percentage of subjects reporting at least 1 unsolicited AE between D0 and D30 was comparable across the 4 study groups: 22.9% (115 / 503) of subjects in Group 1 and 25.7% (129 / 501) of subjects in Group 2; 26.0% (102 / 392) of subjects in Group 3 and 22.6% (67 / 296) of subjects in Group 4. Few subjects reported immediate unsolicited AEs: 0.6% (3 / 503) of subjects in Group 1, 0.2% (1 / 501) of subjects in Group 2, 0.8% (3 / 392) of subjects in Group 3, and 0.7% (2 / 296) of subjects in Group 4. There were no immediate SAEs, including any hypersensitivity events or life-threatening events. On Days 23-37, 9 subjects reported 12 immediate unsolicited AEs. One subject reported 1 immediate unsolicited AE 6 months after D0 vaccination.

[0588] The percentage of subjects reporting at least 1 unsolicited non-serious injection site AR after D0 vaccination was comparable between Groups 1 and 2: 1.4% (7 / 503) and 1.6% (8 / 501), respectively; the percentage of subjects reporting at least 1 unsolicited non-serious injection site AR was numerically higher in Group 3 than in Group 4: 4.3% (17 / 392) and 2.0% (6 / 296), respectively. The most commonly reported unsolicited injection site reaction was pruritus reported in 14 subjects, followed by bruising reported in 13 subjects. The above unsolicited injection site reactions may generally occur after any vaccination.

[0589] In the case of MenACYW-TT conjugate vaccine or The percentages of subjects reporting at least 1 unsolicited non-serious injection site AR at the injection site were comparable: 1.4% (7 / 503) in Group 1, 1.6% (8 / 501) in Group 2, and 1.8% (7 / 392) in Group 3. One subject in Group 2 reported 1 Grade 3 unsolicited non-serious injection site AR (injection site fever) that began on D01, lasted 4 days, and resolved spontaneously. No action was taken. No Grade 3 unsolicited non-serious injection site AR was reported at the injection site for MenACYW-TT conjugate vaccine in Group 1 or in Group 3.

[0590] The percentage of subjects reporting at least 1 unsolicited, nonserious AE within 30 days was comparable across the 4 study groups: 22.7% (114 / 503) of subjects in Group 1, 25.5% (128 / 501) of subjects in Group 2, 26.0% (102 / 392) of subjects in Group 3, and 22.3% (66 / 296) of subjects in Group 4. Infections and infestations were most commonly reported (7.2% [36 / 503] of subjects in Group 1, 8.0% [40 / 501] of subjects in Group 2, 8.2% [32 / 392] of subjects in Group 3, and 6.1% [18 / 296] of subjects in Group 4); the most common type was upper respiratory tract infection.

[0591] Sixteen subjects reported SAEs during the trial; four subjects reported SAEs within 30 days of vaccination on D0. None were considered vaccine-related and none led to study discontinuation. All subjects recovered. No deaths were reported during the study.

[0592] The MenACYW-TT conjugate vaccine was found to be safe for use in adolescents, with no safety concerns identified when given alone or with Tdap and HPV vaccines. The safety profile of the MenACYW-TT conjugate vaccine is similar to that of the licensed Vaccine equivalent.

[0593] f) Phase III clinical trial 1 - Immunogenicity and safety of boosted doses of MenACYW-TT in adolescents and adults

[0594] This study evaluated the safety and immunogenicity of a single dose (10 μg polysaccharide per serogroup, conjugated to a total of 65 μg TT, buffered to pH 6.0 in 0.67% NaCl / 30 mM sodium acetate) of MenACYW-TT given intramuscularly to adolescents (≥15 to <18 years) and adults (18-59 years). Subjects had been given a dose of a quadrivalent meningococcal conjugate vaccine ("priming vaccine") 4-10 years prior to MenACYW-TT (Group 1; n=402).

[0595] Will (Meningococcal (Group A, C, Y and W-135) Capsular Saccharide Diphtheria Toxoid Conjugate Vaccine, referred to herein as "MenACYW-DT", a licensed tetravalent meningococcal conjugate vaccine) was administered to the control group (Group 2; n=407). Subjects in Group 2 were also given a dose of priming vaccine 4-10 years prior.

[0596] In both groups, the primary vaccines were MenACYW-DT (86.3% of all subjects), MenACYW-CRM 197 (11.25% of all subjects; MenACYW-CRM 197 Already discussed above for Clinical Trial 4) or unknown (2.45% of all subjects; 9 in Group 1 and 10 in Group 2). The demographic characteristics of the study groups are shown in Table 17.

[0597]

[0598] hSBA and rabbit serum bactericidal (rSBA) assays were performed 30 days after treatment. hSBA assays were also performed on day 6. The percentage of subjects showing a positive serological response is given in Table 18. For hSBA results, a positive serological response is a post-vaccination titer ≥1:16 when the baseline titer is <1:8, or a 4-fold increase after vaccination when the baseline titer is ≥1:8. 384 subjects in Group 1 and 389 subjects in Group 2 had valid hSBA results on Day 30. For rSBA results, a positive serological response is a post-vaccination titer ≥1:32 when the baseline titer is <1:8, or a 4-fold increase after vaccination when the baseline titer is ≥1:8.

[0599]

[0600]

[0601] The percentage of subjects showing hSBA titers ≥ 1:8 on Day 30 was ≥ 99% for all serogroups, with no significant change in identity to the previously administered vaccine (data not shown).

[0602] Table 19 shows the geometric mean titers (GMT) at day 0 (pre-treatment) and day 30 as determined by hSBA and rSBA. For day 30, hSBA results are presented for each group as a whole and for those who had previously been given MenACYW-DT and MenACYW-CRM. 197 Results for the subgroups of .

[0603]

[0604]

[0605] The results presented above show that for all four serogroups, the percentage of subjects demonstrating a serological response was greater following administration of MenACYW-TT on day 30 after the booster than for MenACYW-DT, as determined using the hSBA. Post-vaccination GMTs according to the hSBA were also numerically higher for the MenACYW-TT vaccine than for MenACYW-DT for all four serogroups.

[0606] In addition, patients who received a single dose of MenACYW-DT or MenACYW-CRM 4 to 10 years ago 197 A single dose of MenACYW-TT was well tolerated in adults and adolescents, and no new safety issues or safety signals were generated. The following items were observed in terms of safety: the occurrence, nature, duration, intensity and relationship to vaccination of any unsolicited systemic AE reported within 30 minutes after vaccination; the occurrence, onset time, day of occurrence, intensity, actions taken and whether the reaction led to early termination of the study for solicited injection site reactions occurring up to 7 days after vaccination; the occurrence, onset time, day of occurrence, intensity, actions taken and whether the reaction led to early termination of the study for systemic reactions occurring up to 7 days after vaccination; the occurrence, nature, onset time, duration, intensity, actions taken, relationship to vaccination (only for systemic AEs) of unsolicited AEs up to D30 (+14 days) and whether the event led to early termination of the study; the occurrence, nature, onset time, duration, severity criteria, relationship to vaccination, results and whether the study led to early termination of SAEs during the entire trial. Solicited AEs were essentially as described above for Phase II clinical trial 4.

[0607] Overall, the percentage of subjects reporting at least 1 solicited reaction was comparable between Groups 1 and 2: 64.3% (256 / 398) of subjects in Group 1 and 65.4% (263 / 402) of subjects in Group 2, respectively. The percentage of subjects reporting at least 1 Grade 3 solicited reaction was comparable between Groups 1 and 2: 5.0% (20 / 398) of subjects in Group 1 and 5.5% (22 / 402) of subjects in Group 2, respectively. The percentage of subjects reporting at least 1 solicited injection site reaction was comparable between Groups 1 and 2: 46.5% (185 / 398) of subjects in Group 1 and 49.3% (198 / 402) of subjects in Group 2, respectively. The most commonly reported solicited injection site reaction was pain, reported by 44.7% (178 / 398) of subjects in Group 1 and 48.8% (196 / 402) of subjects in Group 2. Erythema and swelling were reported less frequently. Erythema and swelling were reported more frequently in Group 1 (5.0% [20 / 398] and 4.0% [16 / 398], respectively) than in Group 2 (1.5% [6 / 402] and 0.7% [3 / 402], respectively). Most were of Grade 1 intensity; the percentages of subjects reporting Grade 2 erythema and swelling were comparable between Group 1 (0.5% [2 / 398] and 0.8% [3 / 398], respectively) and Group 2 (0.2% [1 / 402] and 0.5% [2 / 402], respectively). No Grade 3 erythema or swelling was reported in either group. Most injection site reactions were Grade 1 or 2 in intensity, most began between D0 and D03, and most lasted 1 to 3 days. Few subjects reported Grade 3 solicited injection site reactions: 1.0% (4 / 398) of subjects in Group 1 and 2.0% (8 / 402) of subjects in Group 2 reported Grade 3 pain. No subject in either group reported Grade 3 erythema or swelling.

[0608] The percentage of subjects reporting at least 1 solicited systemic reaction was comparable between Group 1 and Group 2: 55.3% (220 / 398) of subjects in Group 1 and 54.2% (218 / 402) of subjects in Group 2, respectively. Myalgia and headache were the most commonly reported systemic reactions: myalgia was reported by 36.7% (146 / 398) of subjects in Group 1 and 38.8% (156 / 402) of subjects in Group 2; headache was reported by 37.9% (151 / 398) of subjects in Group 1 and 33.3% (134 / 402) of subjects in Group 2. Malaise was reported by 27.6% (110 / 398) of subjects in Group 1 and 26.9% (108 / 402) of subjects in Group 2. Fever was not reported in Group 1 (0.0% [0 / 390]), and was reported by 0.5% (2 / 395) of subjects in Group 2. Overall, most solicited systemic reactions were Grade 1 or 2 in intensity, began between D0 and D30, and lasted for 1 to 3 days. Few subjects reported Grade 3 solicited systemic reactions. The percentages of subjects reporting Grade 3 solicited systemic reactions were similar between Groups 1 and 2: fever (0.0% [0 / 390] and 0.3% [1 / 395], respectively), headache (2.3% [9 / 398] and 3.5% [14 / 402], respectively), malaise (2.8% [11 / 398] and 3.5% [14 / 402], respectively), and myalgia (2.0% [8 / 398] and 2.2% [9 / 402]).

[0609] The percentage of subjects reporting at least 1 unsolicited non-serious AE between D0 and D30 was comparable between Group 1 (26.1% [105 / 402]) and Group 2 (25.3% [103 / 407]). The number of unsolicited non-serious AEs was the same in Group 1 (n=164 AEs) and Group 2 (n=164 AEs). A small but substantial proportion of unsolicited non-serious AEs were considered related to the vaccine administered on D0: 3.0% (12 / 402) of subjects in Group 1 and 2.9% (12 / 407) of subjects in Group 2. Most of these events were Grade 1 or 2 in intensity.

[0610] Two subjects in Group 1 (0.5%) and no subjects in Group 2 (0.0%) reported at least 1 immediate unsolicited AE (dizziness in both subjects).

[0611] A total of 5 subjects in Group 1 (1.2%) and 6 subjects in Group 2 (1.5%) reported at least 1 unsolicited non-serious injection site AR. Injection site bruising was reported by 2 subjects in Group 1 (0.5%) and 3 subjects in Group 2 (0.7%). Injection site pruritus was reported by 2 subjects in Group 1 (0.5%) and 1 subject in Group 2 (0.2%). Injection site fever was reported by 1 subject in Group 1 (0.2%). Injection site discoloration and injection site urticaria were reported by 1 subject each in Group 2 (0.2%). Injection site discoloration was Grade 3 and persisted at the end of the study.

[0612] Infections and infestations were the most commonly reported unsolicited, nonserious systemic AEs (7.5% of subjects [30 / 402] in Group 1 and 6.6% of subjects [27 / 407] in Group 2). Nasopharyngitis was the most commonly reported type of infection or infestation, reported by 1.7% (7 / 402) of subjects in Group 1 and 1.7% (7 / 407) of subjects in Group 2; and upper respiratory tract infections were reported by 1.0% (4 / 402) of subjects in Group 1 and 1.7% (7 / 407) of subjects in Group 2. Also frequently reported in the SOC were respiratory, thoracic, and mediastinal disorders (including cough and oropharyngeal pain): 6.0% (24 / 402) of subjects in Group 1 and 6.6% (27 / 407) of subjects in Group 2. Most unsolicited, nonserious AEs within 30 days of vaccination were Grade 1 or 2 in intensity. The percentage of subjects reporting at least 1 Grade 3 unsolicited non-serious systemic AE was comparable between the two groups: 3.7% (15 / 402) of subjects in Group 1 and 4.2% (17 / 407) of subjects in Group 2.

[0613] Unsolicited non-serious systemic ARs were reported by 2.2% (9 / 402) of subjects in Group 1 and 1.7% (7 / 407) of subjects in Group 2, most commonly reported in the category of systemic disorders and administration site reactions. Fatigue was reported by 0.5% (2 / 402) of subjects in Group 1 and 0.2% (1 / 407) of subjects in Group 2, and was the most commonly reported unsolicited non-serious systemic AR in this category. Only 1 subject in Group 2 reported 2 Grade 3 unsolicited non-serious systemic ARs: Grade 3 nausea in the SOC gastrointestinal disorders and Grade 3 fatigue in the category of systemic disorders and administration site reactions. Both started on D0 and lasted for 2 days. Nausea was treated with medication; no action was taken for fatigue.

[0614] There were no AEs or ARs leading to study discontinuation in any group. Three subjects experienced SAEs within the first 30 days after vaccination: bilateral pulmonary embolism in one subject in Group 1 and major depression and chest pain in one subject each in Group 2. None of these SAEs were considered by the investigator to be related to the vaccine and none led to study discontinuation.

[0615] After D30 and 6 months of follow-up, 6 subjects (4 in Group 1 and 2 in Group 2) experienced a total of 6 SAEs: 4 subjects (1.0%) in Group 1 experienced 4 SAEs, and 2 subjects (0.5%) in Group 2 experienced 2 SAEs. None of these SAEs were considered to be related to the vaccine, and none led to study discontinuation. No deaths were reported during the study.

[0616] The booster dose of MenACYW conjugate vaccine was found to be safe in adolescents and adults aged ≥ 15 years who were primed with quadrivalent meningococcal conjugate vaccine, and no safety concerns were identified. Overall, the safety profile of MenACYW conjugate vaccine is consistent with that of licensed Vaccine equivalent.

[0617] 7. Stability of MenACYW-TT liquid formulation

[0618] Existing ones such as MenACWY-CRM 197 / (tetravalent conjugate with CRM197, where the MenA conjugate was lyophilized) and MCV4-TT Some or all components of meningococcal polysaccharide conjugate vaccines such as ANTIBODYNAMIC® (a completely lyophilized tetravalent conjugate with TT) cannot be stored for long periods of time as liquid formulations. (tetravalent conjugate with TT) is stored as a liquid formulation but has a shelf life of 24 months. In a recent publication, Beresford et al characterised the stability of various existing vaccines and observed deaggregation and loss of immunogenicity under certain conditions, so they suggest that “for any newly developing MenACWY saccharide-protein conjugate vaccines, a key recommendation would be to consider the lyophilization of final product to prevent deleterious degradation that would affect immunogenicity”. Beresford et al, Vaccine 2017 Jun 16;35(28):3598-3606 Abstract.

[0619] Liquid formulations have advantages, at least in that they do not require a lyophilization step during manufacture, do not require a reconstitution step prior to administration, and avoid the risk of possible errors associated with reconstitution. Of course, these advantages are irrelevant if the product itself undergoes degradation that impairs immunogenicity. It would therefore be desirable for a MenACYW conjugate vaccine to be stable for more than 24 months as a liquid formulation. In accordance with the present disclosure, the stability of MenACYW-TT is characterized as follows.

[0620] MenACYW-TT was stored in 0.67% NaCl / 30 mM sodium acetate buffered at pH 6.0 at 2°C-8°C for 54 months and tested for stability at time points selected from 0, 1, 3, 6, 9, 12, 18, 24, 30, 36, 42, 48 and 54 months (see Table 20). Parameters measured included visual appearance, sterility, absence of unusual toxicity, total polysaccharide per serogroup, % free polysaccharide, molecular weight, polydispersity, pH and immunogenicity.

[0621] Accelerated stability testing of MenACYW-TT was also conducted by storing the formulations at 23° C.-27° C. Testing was conducted at one or more of 1 week, 3 months, and 6 months (see Table 21).

[0622] Regarding visual appearance, the solution remained clear and there were no noted defects over the entire time period of 54 months at 2°C-8°C and 6 months at 23°C-27°C (all time points listed above were tested).

[0623] The absence of unusual toxicity was determined by observing rodents (mice or guinea pigs) after one dose of MenACYW-TT. In all tests, all animals survived the 28-day post-dose test period; there were no nonspecific or unexpected responses; and the animals did not lose weight during the test period. Unusual toxicity testing was performed at 0, 12, 24, 36, 42, 48, and 54 month time points.

[0624] The pH was 6.1 at 0 months, and all measurements were within the range of 6.1 to 6.3 over the entire time course (time points for pH were 0, 1, 6, 12, 24, 36, 42, 48, and 54 months at 2°C-8°C and 6 months at 23°C-27°C). No growth was observed at any time point tested, including 54 months (time points for sterility were 0, 12, 24, 36, 42, 48, and 54 months at 2°C-8°C and 6 months at 23°C-27°C).

[0625] By Dionex TM Chromatographic method to measure total polysaccharides. TM % free polysaccharides were measured by size exclusion chromatography / multi-angle light scattering (SEC / MALS).

[0626] In Table 20 are the results for 2°C-8°C, and in Table 21 are the results for 23°C-27°C.

[0627] Table 20: Stability test results stored at 2°C to 8°C.

[0628]

[0629]

[0630] NS: Not scheduled. Prior to testing, serum samples were stored at a temperature < -20°C where they are believed to remain stable for approximately 6-7 months.

[0631] Table 21: Stability test results stored at 23°C to 27°C.

[0632]

[0633] NS: Not scheduled.

[0634] The results indicate good stability throughout the period of 54 months at 2°C-8°C and 6 months at 23°C-27°C. In particular, at 2°C-8°C, for all four serogroups, there was minimal loss of conjugation of the polysaccharide to TT (% free polysaccharide increased by 0-3%), and immunogenicity was essentially maintained at all time points except 24 months, where the low values ​​appear to be outliers and / or result from technical issues based on observations at 36 months and beyond, which were more similar to the values ​​measured at 0 and 12 months. At 23°C-27°C, free polysaccharide increased at 6 months for MenA, MenY and MenW, but levels were still below the acceptable limit of 40%. Immunogenicity was still present at six months.

[0635] Based on these results, we can conclude that MenACYW-TT can be maintained as a liquid formulation for long periods of time under refrigeration, such as during packaging, distribution and storage, and does not require lyophilization or other preservation measures.

[0636] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

[0637] In summary, the present invention also includes the following items:

[0638] 1. A Neisseria meningitidis vaccine composition comprising:

[0639] a) a first conjugate of MenA capsular polysaccharide and a carrier protein;

[0640] b) a second conjugate of MenC capsular polysaccharide and a carrier protein;

[0641] c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and

[0642] d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein:

[0643] (i) the second conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide, and the O-acetylation level of the polysaccharide of the second conjugate is 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide, and wherein the single-end-linked conjugated polysaccharide has a terminal unlinked sugar;

[0644] (ii) the MenA capsular polysaccharide is attached to the carrier protein via a linker comprising a carbamate, a spacer and an amide, wherein the spacer is between the carbamate and the amide and comprises 2-10 linear carbons, and the polysaccharide to carrier protein mass ratio of the first conjugate is 0.3 to 1.5;

[0645] (iii) the MenC, MenW-135 and MenY capsular polysaccharides are attached to the carrier protein via secondary amines and at least one of the conjugates has a weight average molecular weight ranging from 300 kDa to 1500 kDa;

[0646] (iv) the carrier protein is tetanus toxoid, and

[0647] The composition comprises less than 20% by weight of free polysaccharides relative to total polysaccharides.

[0648] 2. A Neisseria meningitidis vaccine composition according to claim 1, wherein the first, second, third and / or fourth conjugate is a population comprising molecules with a molecular weight ranging from 700 kDa to 1400 kDa or from 800 kDa to 1300 kDa.

[0649] 3. A Neisseria meningitidis vaccine composition comprising a conjugate of MenC capsular polysaccharide and a carrier protein, wherein the conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide both attached to the carrier protein via a secondary amine, and the O-acetylation level of the polysaccharide of the conjugate of MenC capsular polysaccharide to the carrier protein ranges from 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide, wherein the carrier protein is tetanus toxoid.

[0650] 4. The Neisseria meningitidis vaccine composition according to item 3, wherein the conjugate of the MenC capsular polysaccharide and the carrier protein:

[0651] a) having a weight average molecular weight ranging from 300 kDa to 1500 kDa; or

[0652] b) is a population comprising molecules with a molecular weight in the range of 700 kDa to 1400 kDa or 800 kDa to 1300 kDa.

[0653] 5. The Neisseria meningitidis vaccine composition according to any one of items 1 to 4, wherein the Neisseria meningitidis vaccine composition does not contain an adjuvant and / or comprises an acetate buffer having a pH of 5.5 to 6.5.

[0654] 6. A Neisseria meningitidis vaccine composition according to any one of items 1 to 5, wherein the degree of O-acetylation of the MenC capsular polysaccharide ranges from 0.6 to 1.5 μmol / mg polysaccharide or from 0.8 to 1.4 μmol / mg polysaccharide.

[0655] 7. A Neisseria meningitidis vaccine composition according to any one of items 1 to 6, wherein the conjugate comprising MenC polysaccharide comprises one or more modifications selected from the group consisting of: (i) a primary hydroxyl group at position 7, (ii) a (2-hydroxy)ethoxy group at the reducing end, and (iii) conjugation to the carrier protein, wherein the modifications are present at not less than 25 nmol / mg polysaccharide.

[0656] 8. The Neisseria meningitidis vaccine composition according to any one of items 1 to 7, wherein the size of the MenC polysaccharide is reduced by 3-8 times or by 1 / 3-1 / 8 relative to the native MenC polysaccharide.

[0657] 9. The Neisseria meningitidis vaccine composition according to any one of items 1, 2 and 5-8, wherein:

[0658] (i) the mass ratio of MenA capsular polysaccharide to carrier protein of the first conjugate is 0.5 to 1.5 or 0.7 to 1.4;

[0659] (ii) the mass ratio of MenC capsular polysaccharide to carrier protein of the second conjugate is from 0.3 to 1.1;

[0660] (iii) the mass ratio of MenY capsular polysaccharide to carrier protein of the third conjugate is from 0.3 to 1.1; and / or

[0661] (iv) The mass ratio of MenW-135 capsular polysaccharide to carrier protein of the fourth conjugate is 0.3 to 1.3.

[0662] 10. The Neisseria meningitidis vaccine composition according to item 3 or 4, further comprising: a conjugate of MenA capsular polysaccharide and a carrier protein, wherein the polysaccharide to carrier protein mass ratio is 0.5 to 1.5; a conjugate of MenW-135 capsular polysaccharide and a carrier protein, wherein the polysaccharide to carrier protein mass ratio is 0.3 to 1.3; and a conjugate of MenY capsular polysaccharide and a carrier protein, wherein the polysaccharide to carrier protein mass ratio is 0.5 to 1.3.

[0663] 11. The Neisseria meningitidis vaccine composition according to claim 10, wherein:

[0664] (i) the MenA capsular polysaccharide is attached to the carrier protein via a linker comprising a carbamate, a spacer and an amide, wherein the spacer is between the carbamate and the amide and comprises 2-10 linear carbons, and the conjugate has a MenA capsular polysaccharide to carrier protein mass ratio of 0.3 to 1.5;

[0665] (ii) wherein the MenW-135 and MenY capsular polysaccharides are attached to the carrier protein via secondary amines and the weight average molecular weight of the conjugate ranges from 300 kDa to 1500 kDa;

[0666] (iii) the composition comprises less than 20% by weight of free polysaccharides relative to total polysaccharides.

[0667] 12. The Neisseria meningitidis vaccine composition according to any one of items 1, 2 and 5-11, wherein the MenA capsular polysaccharide is attached to the carrier protein via a linker of formula (I):

[0668]

[0669] Wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein.

[0670] 13. The Neisseria meningitidis vaccine composition according to claim 1, wherein:

[0671] (a) the MenA capsular polysaccharide is attached to a tetanus toxoid carrier protein via a linker of formula (I):

[0672]

[0673] wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein, and wherein the mass ratio of the MenA capsular polysaccharide to the tetanus toxoid carrier protein is from 0.5 to 1.5; and / or

[0674] (b) the MenC capsular polysaccharide is attached to a tetanus toxoid carrier protein via a secondary amine, wherein the second conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide, wherein the O-acetylation level of the polysaccharide is from 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide, wherein the weight average molecular weight of the second conjugate ranges from 300 kDa to 1500 kDa, wherein the size of the MenC capsular polysaccharide is reduced by 3-8 times or by 1 / 3-1 / 8 relative to native MenC polysaccharide, and wherein the mass ratio of the MenC capsular polysaccharide to the tetanus toxoid carrier protein is from 0.3 to 1.1; and / or

[0675] (c) the MenW-135 capsular polysaccharide is attached to a tetanus toxoid carrier protein via a secondary amine, and wherein the mass ratio of the MenW-135 capsular polysaccharide to the tetanus toxoid carrier protein is 0.3 to 1.3; and / or

[0676] (d) wherein the MenY capsular polysaccharide is attached to the tetanus toxoid carrier protein via a secondary amine, and wherein the mass ratio of the MenY capsular polysaccharide to the tetanus toxoid carrier protein is from 0.5 to 1.3; and

[0677] The composition comprises less than 20% by weight of free polysaccharides relative to total polysaccharides.

[0678] 14. The Neisseria meningitidis vaccine composition according to any one of items 1 to 13, for use in a method of vaccinating a subject against Neisseria meningitidis,

[0679] wherein the vaccine is preferably administered intramuscularly, and / or

[0680] The subject is preferably aged between 6 weeks and 3 years old or 50 years old or older.

Claims

1. A Neisseria meningitidis vaccine composition comprising: a) a first conjugate of MenA capsular polysaccharide and a carrier protein; b) a second conjugate of MenC capsular polysaccharide and a carrier protein; c) a third conjugate of MenW-135 capsular polysaccharide and a carrier protein; and d) a fourth conjugate of MenY capsular polysaccharide and a carrier protein; wherein: (i) the second conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide, the O-acetylation level of the polysaccharide of the second conjugate is from 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide, the single-end-linked conjugated polysaccharide has a terminal unlinked sugar, and the second conjugate has a MenC capsular polysaccharide to carrier protein mass ratio of 0.3 to 1.1; (ii) the MenA capsular polysaccharide is attached to the carrier protein via a linker comprising a residue of adipic acid dihydrazide, the first conjugate has a MenA capsular polysaccharide to carrier protein mass ratio of 0.7 to 1.4, and the first conjugate has a weight average molecular weight range of 300 kDa to 1500 kDa; (iii) the third conjugate has a MenW-135 capsular polysaccharide to carrier protein mass ratio of 0.3 to 1.3; (iv) the fourth conjugate has a MenY capsular polysaccharide to carrier protein mass ratio of 0.3 to 1.1; (v) the MenC, MenW-135 and MenY capsular polysaccharides are directly attached to the carrier protein via secondary amines and the weight average molecular weight of each of the conjugates ranges from 300 kDa to 1500 kDa; (vi) the carrier protein is tetanus toxoid, and The composition comprises less than 20% by weight of free polysaccharides relative to total polysaccharides.

2. The Neisseria meningitidis vaccine composition according to claim 1, wherein the first, second, third and / or fourth conjugate is a population comprising molecules with a molecular weight ranging from 700 kDa to 1400 kDa.

3. The Neisseria meningitidis vaccine composition according to claim 2, wherein the first, second, third and / or fourth conjugate is a population comprising molecules with a molecular weight ranging from 800 kDa to 1300 kDa.

4. A Neisseria meningitidis vaccine composition comprising a conjugate of a MenC capsular polysaccharide and a carrier protein, wherein the conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide both attached to the carrier protein via secondary amines, the O-acetylation level of the polysaccharide of the conjugate of the MenC capsular polysaccharide and the carrier protein ranges from 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide, and the weight average molecular weight of the conjugate of the MenC capsular polysaccharide and the carrier protein ranges from 300 kDa to 1500 kDa; wherein the carrier protein is tetanus toxoid.

5. The Neisseria meningitidis vaccine composition according to claim 4, wherein the conjugate of the MenC capsular polysaccharide and the carrier protein is a population comprising molecules with a molecular weight ranging from 700 kDa to 1400 kDa.

6. The Neisseria meningitidis vaccine composition according to claim 5, wherein the conjugate of the MenC capsular polysaccharide and the carrier protein is a population comprising molecules with a molecular weight ranging from 800 kDa to 1300 kDa.

7. The Neisseria meningitidis vaccine composition according to any one of claims 1 to 6, wherein the Neisseria meningitidis vaccine composition does not contain an adjuvant and / or comprises an acetate buffer having a pH of 5.5 to 6.

5.

8. The Neisseria meningitidis vaccine composition according to any one of claims 1 to 6, wherein the degree of O-acetylation of the MenC capsular polysaccharide ranges from 0.6 to 1.5 μmol / mg polysaccharide.

9. The Neisseria meningitidis vaccine composition according to claim 8, wherein the degree of O-acetylation of the MenC capsular polysaccharide ranges from 0.8 to 1.4 μmol / mg polysaccharide.

10. The Neisseria meningitidis vaccine composition according to any one of claims 1 to 6, wherein the conjugate comprising MenC polysaccharide comprises one or more modifications selected from the group consisting of: (i) a primary hydroxyl group at position 7, (ii) a (2-hydroxy)ethoxy group at the reducing end, and (iii) conjugation to the carrier protein, wherein the modifications are present at not less than 25 nmol / mg polysaccharide.

11. The Neisseria meningitidis vaccine composition according to any one of claims 1 to 6, wherein the size of the MenC polysaccharide is reduced by 3-8 times relative to the native MenC polysaccharide.

12. The Neisseria meningitidis vaccine composition according to any one of claims 1 to 3, wherein the MenA capsular polysaccharide is attached to the carrier protein via a linker of formula (I): Wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein.

13. The Neisseria meningitidis vaccine composition according to claim 1, wherein: (a) The MenA capsular polysaccharide is attached to a tetanus toxoid carrier protein via a linker of formula (I): wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein, and wherein the mass ratio of the MenA capsular polysaccharide to the tetanus toxoid carrier protein is from 0.5 to 1.5; and / or (b) the MenC capsular polysaccharide is attached to a tetanus toxoid carrier protein via a secondary amine, wherein the second conjugate is a population comprising a double-end-linked conjugated polysaccharide and a single-end-linked conjugated polysaccharide, wherein the O-acetylation level of the polysaccharide is from 0.3 μmol / mg polysaccharide to 1.6 μmol / mg polysaccharide, wherein the weight average molecular weight of the second conjugate ranges from 300 kDa to 1500 kDa, wherein the size of the MenC capsular polysaccharide is reduced by 3-8 times relative to native MenC polysaccharide, and wherein the mass ratio of the MenC capsular polysaccharide to the tetanus toxoid carrier protein is from 0.3 to 1.1; and / or (c) the MenW-135 capsular polysaccharide is attached to a tetanus toxoid carrier protein via a secondary amine, and wherein the mass ratio of the MenW-135 capsular polysaccharide to the tetanus toxoid carrier protein is 0.3 to 1.3; and / or (d) wherein the MenY capsular polysaccharide is attached to the tetanus toxoid carrier protein via a secondary amine, and wherein the mass ratio of the MenY capsular polysaccharide to the tetanus toxoid carrier protein is from 0.5 to 1.3; as well as The composition comprises less than 20% by weight of free polysaccharides relative to total polysaccharides.

14. The Neisseria meningitidis vaccine composition according to any one of claims 1 to 6 and 13, for use in a method of vaccinating a subject with a Neisseria meningitidis vaccine, Wherein the vaccine is administered intramuscularly.

15. The Neisseria meningitidis vaccine composition according to any one of claims 1 to 6 and 13, for use in a method of vaccinating a subject with a Neisseria meningitidis vaccine, wherein the subject is aged between 6 weeks and 3 years old or 50 years old or older.

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