Neisseria Meningitidis Vaccine

1 . A Neisseria meningitidis vaccine composition comprising:

a) a first conjugate of MenA capsular polysaccharide to a carrier protein;

b) a second conjugate of MenC capsular polysaccharide to a carrier protein;

c) a third conjugate of MenW-135 capsular polysaccharide to a carrier protein; and

d) a fourth conjugate of MenY capsular polysaccharide to a carrier protein;

wherein the second conjugate is a population comprising double-end-linked conjugated polysaccharides and single-end-linked conjugated polysaccharides which both are attached to the carrier protein through a secondary amine, and the polysaccharides of the second conjugate have an O-acetylation level of 0.3 μmol/mg polysaccharide to 1.6 μmol/mg polysaccharide.

2 . A Neisseria meningitidis vaccine composition comprising:

a) a first conjugate of MenA capsular polysaccharide to a carrier protein;

b) a second conjugate of MenC capsular polysaccharide to a carrier protein;

c) a third conjugate of MenW-135 capsular polysaccharide to a carrier protein; and

d) a fourth conjugate of MenY capsular polysaccharide to a carrier protein;

wherein the second conjugate is a population comprising single-end-linked conjugated polysaccharides which are attached to the carrier protein through a secondary amine, wherein the single-end-linked conjugated polysaccharides have a terminal unlinked saccharide, wherein the terminal saccharide has a primary hydroxyl or secondary amine linkage at the 7 position, or wherein the reducing end is modified with a (2-hydroxy)ethoxy or secondary amine linkage.

3 . A Neisseria meningitidis vaccine composition comprising:

a) a first conjugate of MenA capsular polysaccharide to a carrier protein;

b) a second conjugate of MenC capsular polysaccharide to a carrier protein;

c) a third conjugate of MenW-135 capsular polysaccharide to a carrier protein; and

d) a fourth conjugate of MenY capsular polysaccharide to a carrier protein;

wherein the MenA capsular polysaccharide is attached to the carrier protein through 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 first conjugate has a polysaccharide to carrier protein mass ratio of 0.3 to 1.5.

4 . A Neisseria meningitidis vaccine composition comprising:

a) a first conjugate of MenA capsular polysaccharide to a carrier protein;

b) a second conjugate of MenC capsular polysaccharide to a carrier protein;

c) a third conjugate of MenW-135 capsular polysaccharide to a carrier protein; and

d) a fourth conjugate of MenY capsular polysaccharide to a carrier protein;

wherein the MenA capsular polysaccharide is attached to the carrier protein through 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 through a secondary amine; and

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

5 . A Neisseria meningitidis vaccine composition comprising:

a) a first conjugate of MenA capsular polysaccharide to a carrier protein;

b) a second conjugate of MenC capsular polysaccharide to a carrier protein;

c) a third conjugate of MenW-135 capsular polysaccharide to a carrier protein; and

d) a fourth conjugate of MenY capsular polysaccharide to a carrier protein;

wherein the carrier protein is tetanus toxoid;

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

6 . A Neisseria meningitidis vaccine composition comprising:

a) a first conjugate of MenA capsular polysaccharide to a carrier protein;

b) a second conjugate of MenC capsular polysaccharide to a carrier protein;

c) a third conjugate of MenW-135 capsular polysaccharide to a carrier protein; and

d) a fourth conjugate of MenY capsular polysaccharide to a carrier protein;

wherein the carrier protein is tetanus toxoid;

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

7 . The vaccine composition of any one of the preceding claims , wherein the first, second, third, and/or fourth conjugates are a population comprising molecules with a molecular weight in the range of 700 kDa to 1400 kDa or 800 kDa to 1300 kDa.

8 . A Neisseria meningitidis vaccine composition comprising a conjugate of MenC capsular polysaccharide to a carrier protein, wherein the conjugate is a population comprising double-end-linked conjugated polysaccharides and single-end-linked conjugated polysaccharides which both are attached to the carrier protein through a secondary amine, and the polysaccharides of the conjugate of MenC capsular polysaccharide to the carrier protein have an O-acetylation level ranging from 0.3 μmol/mg polysaccharide to 1.6 μmol/mg polysaccharide.

9 . The vaccine composition of claim 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 having a molecular weight in the range of 700 kDa to 1400 kDa or 800 kDa to 1300 kDa.

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

11 . The vaccine composition of any one of the preceding claims , wherein the MenC polysaccharide has a degree of O-acetylation ranging from 0.6 to 1.5 μmol/mg polysaccharide or 0.8 to 1.4 μmol/mg polysaccharide.

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.

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.

14 . The vaccine composition of any one of the preceding claims , wherein the conjugate comprising MenC polysaccharide is a population comprising double-end-linked conjugated polysaccharides and single-end-linked conjugated polysaccharides.

15 . The vaccine composition of claim 14 , wherein the single-end-linked polysaccharides of the second conjugate comprise a terminal unlinked saccharide, wherein the single-end-linked conjugated polysaccharides have a terminal unlinked saccharide, wherein the terminal saccharide has a primary hydroxyl at the 7 position, or wherein the reducing end is modified with a (2-hydroxy)ethoxy.

16 . The vaccine composition of any one of the preceding claims , wherein the conjugate comprising MenC polysaccharide comprises one or more modifications chosen from (i) a primary hydroxyl at the 7 position, (ii) a (2-hydroxy)ethoxy at the reducing end, and (iii) a conjugation to the carrier protein, wherein the modifications are present at no less than 25 nmol/mg polysaccharide.

17 . The vaccine composition of any one of the preceding claims , comprising a conjugate of MenW-135 and/or MenY polysaccharide which comprises one or more modifications chosen from (i) a primary hydroxyl at a position of a vicinal diol in a native MenW-135 or MenY polysaccharide and (ii) a conjugation to the carrier protein, wherein the modifications are present at no less than 60 nmol/mg polysaccharide.

18 . The vaccine composition of claim 16 or 17 , wherein the modifications are present in an amount 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.

19 . The vaccine composition of any one of the preceding claims , wherein the MenC polysaccharide is reduced in size by 3×-8× relative to native MenC polysaccharide.

20 . The vaccine composition of any one of the preceding claims , comprising a conjugate of MenA capsular polysaccharide to a carrier protein having a polysaccharide to carrier protein mass ratio of 0.5 to 1.5 or 0.7 to 1.4.

21 . The vaccine composition of claim 20 , wherein the MenA conjugate has a polysaccharide to carrier protein mass ratio of 0.8 to 1.3.

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

23 . The vaccine composition of claim 22 , wherein the MenC conjugate has a polysaccharide to carrier protein mass ratio of 0.4 to 0.8.

24 . The vaccine composition of any one of the preceding claims , comprising a conjugate of MenW-135 capsular polysaccharide to a carrier protein having a polysaccharide to carrier protein mass ratio of 0.3 to 1.3.

25 . The vaccine composition of claim 24 , wherein the MenW-135 conjugate has a polysaccharide to carrier protein mass ratio of 0.6 to 1.3.

26 . The vaccine composition any one of the preceding claims , comprising a conjugate of MenY capsular polysaccharide to a carrier protein having a polysaccharide to carrier protein mass ratio of 0.5 to 1.3.

27 . The vaccine composition of claim 26 , wherein the MenY conjugate has a polysaccharide to carrier protein mass ratio of 0.5 to 0.9.

28 . The vaccine composition of any one of the preceding claims , wherein the composition comprises less than 20% free polysaccharide by weight.

29 . The vaccine composition of claim 28 , wherein the composition comprises less than 10% free polysaccharide by weight, less than 5% free polysaccharide by weight, or substantially lacks free polysaccharide.

30 . The vaccine composition of any one of the preceding claims , wherein the polysaccharide of the MenA, MenC, MenW-135, or MenY conjugate is attached to the carrier protein through a linker.

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

32 . The vaccine composition of claims 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 saccharide repeat units.

33 . The vaccine composition of claims 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 saccharide repeat units.

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

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

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

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

wherein PS indicates attachment to the polysaccharide and PR indicates attachment to the carrier protein.

38 . The vaccine composition of any one of claims 30-37 , wherein the linker is in the MenA conjugate.

39 . The vaccine composition of any one of claims 30-37 , wherein the linker is in the MenC conjugate.

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

41 . The vaccine composition of any one of claims 30-37 , wherein the linker is in the MenY conjugate.

42 . The vaccine composition of any one of the preceding claims , wherein the polysaccharide of the MenA, MenC, MenW-135, and/or MenY conjugate is attached to the 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.

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

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

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

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

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

48 . The vaccine composition of any one of the preceding claims , wherein the carrier protein is tetanus toxoid.

49 . A method of producing a conjugate of a Neisseria meningitidis capsular polysaccharide to a carrier protein, comprising:

a) activating the polysaccharide with an activating agent that can form a carbamate linkage wherein the activating agent is present in a molar excess over the polysaccharide of 20-fold to 50-fold;

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

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

50 . The method of claim 49 , wherein the dihydrazide linker is added at a mole ratio of 0.4 to 0.6 relative to polysaccharide repeat units.

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

52 . The method of any one of claims 49 to 51 , wherein the dihydrazide linker is adipic acid dihydrazide.

53 . The method of any one of claims 49 to 51 , wherein the derivatized polysaccharide is at a starting concentration of 10 g/L to 20 g/L in the conjugation reaction.

54 . The method of any one of claims 49 to 51 , wherein the activating agent comprises a carbonyl bound to two N-linked heteroaryls such as CDI (1,1′-Carbonyldiimidazole) and CDT (1,1′-Carbonyl-di-(1,2,4-triazole), or other appropriate leaving groups.

55 . The method of claim 54 , wherein the activating agent is carbonyl diimidazole.

56 . The method of any one of claims 49 to 51 , wherein the activating agent is present in the activating step in a molar excess over the polysaccharide of 35-fold to 45-fold.

57 . The method of any one of claims 49 to 51 , wherein the conjugating step comprises reacting the carrier protein with N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide.

58 . The method of any one of claims 49 to 51 , wherein the polysaccharide is MenA capsular polysaccharide.

59 . The method of any one of claims 49 to 51 , wherein the polysaccharide is MenC capsular polysaccharide.

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

61 . A method of producing a conjugate of a Neisseria meningitidis capsular polysaccharide to a carrier protein, comprising:

a) partially de-O-acetylating the polysaccharide by alkaline hydrolysis;

b) activating the polysaccharide by periodate treatment, thereby converting diols to aldehydes to an extent of at least 20 nmol aldehyde per mg polysaccharide;

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

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

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%.

64 . The method of any one of claims 61 to 63 , wherein following de-O-acetylation, the polysaccharide has a degree of O-acetylation from 0.6 μmol/mg polysaccharide to 1.5 μmol/mg polysaccharide or 0.8 to 1.4 μmol/mg polysaccharide.

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.

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.

67 . The method of any one of claims 61 to 66 , wherein the activated polysaccharide is at a starting concentration of 20 g/L to 50 g/L in the conjugation reaction.

68 . The method of any one of claims 61 to 66 , wherein the polysaccharide is MenC capsular polysaccharide.

69 . The method of any one of claims 61 to 66 , wherein the polysaccharide is MenA capsular polysaccharide.

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

71 . The method of any one of claims 61 to 66 , wherein the polysaccharide is reduced in size to 30 to 150 kDa or to 50 to 100 kDa before the conjugation reaction.

72 . A method of producing a conjugate of a Neisseria meningitidis capsular polysaccharide to a carrier protein, comprising:

a) activating the polysaccharide by periodate treatment, thereby converting diols to aldehydes to an extent of at least 50 nmol aldehyde per mg polysaccharide;

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

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

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

75 . The method of any one of claims 72 and 73 , wherein the polysaccharide is MenC capsular polysaccharide.

76 . The method of any one of claims 72 and 73 , wherein the polysaccharide is MenA capsular polysaccharide.

77 . The method of any one of claims 72 to 76 , wherein the polysaccharide is reduced in size to 100 to 200 kDa or to 125 to 175 kDa before the conjugation reaction.

78 . The method of any one of claims 49, 61, and 72 , wherein the polysaccharide is reduced in size by acid hydrolysis and/or heat.

79 . The method of any one of claims 49, 61, and 72 , wherein the polysaccharide is reduced in size by oxidative cleavage.

80 . The method of any one of claims 72 to 79 , wherein reductive amination comprises reducing imines to amines using a cyanoborohydride, or other reducing reagents such as pyridine borane (C 5 H 8 BN) and picoline borane complex (C 6 H 7 N·BH 3 ).

81 . The method of any one of claims 72 to 79 , further comprising converting unreacted aldehydes in the conjugate to alcohols with a reducing reagent.

82 . The method of claim 81 , wherein the reducing reagent is a borohydride.

83 . The method of any one of claims 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.

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

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

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

a) contacting a hydrophobic interaction chromatography resin with the mixture, wherein the conjugate binds the resin;

b) eluting free polysaccharide from the resin; and

c) eluting the conjugate from the resin with an aqueous liquid, wherein the aqueous liquid is free of salt or contains less salt than the mixture, thereby obtaining a composition comprising purified conjugate.

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

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 0.8 to 1.2 M.

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

90 . The method of any one of claims 86 to 89 , wherein the aqueous liquid is water.

91 . The method of any one of claims 86 to 90 , wherein the composition comprises less than 20% free polysaccharide by weight, less than 10% free polysaccharide by weight, less than 5% free polysaccharide by weight, or substantially lacks free polysaccharide.

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

93 . The method of any one of claims 49 to 92 , wherein the carrier protein is tetanus toxoid.

94 . A conjugate produced according to the method of any one of claims 49 to 85 .

95 . A vaccine composition comprising:

a) a first conjugate of MenA capsular polysaccharide to a carrier protein;

b) a second conjugate of MenC capsular polysaccharide to a carrier protein;

c) a third conjugate of MenW-135 capsular polysaccharide to a carrier protein; and

d) a fourth conjugate of MenY capsular polysaccharide to a carrier protein;

wherein one, two, three, or all of the first, second, third, and fourth conjugates was produced according to the method of any one of claims 49 to 85 .

96 . The vaccine composition of any one of claims 1-48 or 95 , which is free of adjuvant.

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

98 . The vaccine composition of claim 97 , comprising acetate buffer with a pH of 5.5 to 6.5.

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

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

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

102 . The vaccine composition of any one of claims 1-48 or 95-101 , wherein at least one, two, three, or all four of the first, second, third, and fourth conjugates comprise multiple points of attachment between the polysaccharides and the carrier proteins.

103 . The vaccine composition of any one of claims 1-48 or 95-102 , formulated for intramuscular administration.

104 . A single unit dose of the vaccine composition of any one of claims 1 - 48 or 9495 - 103 , comprising from 6 μg to 15 μg of each of the MenA, MenC, MenW-135, and MenY polysaccharides.

105 . The single unit dose of claim 104 , wherein the carrier protein is present in an amount from 50 μg to 80 μg.

106 . The single unit dose of claim 104 or 105 , which is contained in a syringe.

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

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

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

110 . Use of the vaccine composition of any one of claims 1-48 or 95-103 or the single unit dose of any one of claims 104-108 to immunize a subject against Neisseria meningitidis.

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

112 . The method or use of any one of claims 109-111 , wherein the vaccine is administered intramuscularly.

113 . The method or use of any one of claims 109-112 , wherein the subject is age 6 weeks to 3 years.

114 . The method of claim 113 , wherein the subject is 2 months, 6 months, 12 months, or 15 months of age.

115 . The method or use of any one of claims 109-112 , wherein the subject is age 50 years or more, 55 years or more, 60 years or more, or 65 years or more.

116 . The method or use of any of claims 109-115 , wherein the vaccine is administered as a 0.5 mL dose.

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

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

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

120 . The method or use of any of claims 109-119 , further comprising administering a vaccine that is not directed to Neisseria meningitidis at the same time as, but not in the same injection as, the MenACYW-TT vaccine.

121 . The method or use of claim 120 , wherein non- Neisseria meningitidis vaccine is directed to preventing varicella, diphtheria, Hib, hepatitis b, measles, mumps, pertussis, polio, pneumococcus, rotavirus, rubella, or tetanus infections.

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

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

124 . The method or use of claim 123 , wherein the subject received the Neisseria meningitidis capsular saccharide conjugate vaccine four months to ten years earlier.

125 . The method or use of any one of claims 109-122 , wherein the subject did not previously receive a Neisseria meningitidis capsular saccharide conjugate vaccine.