Adjuvanted formulations of booster vaccines

Abstract

The invention improves TdaP vaccines by including a TLR agonist in them. This agonist can provide stronger protection, longer-lasting protection, and/or can reduce the amount of antigen which is required to achieve a particular immune response.

Claims

1. An immunogenic composition comprising a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, an aluminium salt adjuvant, and a Toll-like receptor (TLR) agonist, wherein the immunogenic composition comprises an excess as measured in limit of flocculation (Lf) units of tetanus toxoid relative to diphtheria toxoid.

2. The immunogenic composition of claim 1, wherein the TLR agonist is a TLR4 agonist or a TLR 7 agonist.

3. The immunogenic composition of claim 1, wherein the TLR agonist is adsorbed to the aluminium salt adjuvant.

4. The immunogenic composition of claim 1, wherein 1, 2 or 3 of the toxoids is/are adsorbed to the aluminium salt adjuvant.

5. The immunogenic composition of claim 1, with an Al+++ concentration 0.5 mg/ml.

6. The immunogenic composition of claim 1, wherein the aluminium salt is an aluminium hydroxide.

7. The immunogenic composition of claim 1, wherein the TLR agonist is a compound of formula (K), or a pharmaceutically acceptable salt thereof, Formula (K): ##STR00021## wherein: R.sup.1 is H, C.sub.1-C.sub.6alkyl, C(R.sup.5).sub.2OH, L.sup.1R.sup.5, L.sup.1R.sup.6, L.sup.2R.sup.5, L.sup.2R.sup.6, OL.sup.2R.sup.5, or OL.sup.2R.sup.6; L.sup.1 is C(O)or O; L.sup.2 is C.sub.1-C.sub.6alkylene, C.sub.2-C.sub.6alkenylene, arylene, heteroarylene or ((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p, wherein the C.sub.1-C.sub.6alkylene and C.sub.2-C.sub.6alkenylene of L.sup.2 are optionally substituted with 1 to 4 fluoro groups; each L.sup.3 is independently selected from C.sub.1-C.sub.6alkylene and ((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p, wherein the C.sub.1-C.sub.6alkylene of L.sup.3 is optionally substituted with 1 to 4 fluoro groups; L.sup.4 is arylene or heteroarylene; R.sup.2 is H or C.sub.1-C.sub.6alkyl; R.sup.3 is selected from C.sub.1-C.sub.4alkyl, L.sup.3R.sup.5, L.sup.1R.sup.5, L.sup.3R.sup.7, L.sup.3L.sup.4L.sup.3R.sup.7, L.sup.3L.sup.4R.sup.5, L.sup.3L.sup.4L.sup.3R.sup.5, OL.sup.3R.sup.5, OL.sup.3R.sup.7, OL.sup.3L.sup.4R.sup.7, OL.sup.3L.sup.4L.sup.3R.sup.7, OR.sup.8, OL.sup.3L.sup.4R.sup.5, OL.sup.3L.sup.4L.sup.3R.sup.5 and C(R.sup.5).sub.2OH; each R.sup.4 is independently selected from H and fluoro; R.sup.5 is P(O)(OR.sup.9).sub.2, R.sup.6 is CF.sub.2P(O)(OR.sup.9).sub.2 or C(O)OR.sup.10; R.sup.7 is CF.sub.2P(O)(OR.sup.9).sub.2 or C(O)OR.sup.10; R.sup.8 is H or C.sub.1C.sub.4alkyl; each R.sup.9 is independently selected from H and C.sub.1-C.sub.6alkyl; R.sup.10 is H or C.sub.1-C.sub.4alkyl; each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or 4.

8. The immunogenic composition of claim 1, wherein the TLR agonist is compound K2, ##STR00022##

9. The immunogenic composition of claim 1, wherein the TLR agonist comprises at least one adsorptive moiety which allows it to adsorb to insoluble metal salts.

10. The immunogenic composition of claim 9, wherein the adsorptive moiety is a phosphate or a phosphonate.

11. The immunogenic composition of claim 1, wherein the TLR agonist has formula (C), (D), (E), (F), (G), (H), (I), (II), (J) or (K) as defined below, or is a 3-0-deacylated monophosphoryl lipid A (3d-MPL), Formulae (C), (D), (E), and (H) are as follows: ##STR00023## wherein: P.sup.3 is selected from H, C.sub.1-C.sub.6alkyl, CF.sub.3, and ((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pO.sub.s and -Y-L-X P(O)(OR.sup.X)(OR.sup.Y); and P.sup.4 is selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkylaryl and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); with the proviso that at least one of P.sup.3 and P.sup.4 is -Y-L-XP(O)(OR.sup.X)(OR.sup.Y), .sup.5 is selected from H, C.sub.1-C.sub.6alkyl, and -Y-L-XP(O)(OR.sup.x)(OR.sup.Y); P.sup.6 is selected from H, C.sub.1-C.sub.6alkyl each optionally substituted with 1 to 3 substituents selected from C.sub.1-C.sub.4alkyl and OH, and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); and P.sup.7 is selected from H, C.sub.1-C.sub.6alkyl, ((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pO.sub.s, NHC.sub.1-C.sub.6alkyl and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); with the proviso that at least one of P.sup.5, P.sup.6 and P.sup.7 is -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); P.sup.8 is selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, NHC.sub.1-C.sub.6alkyl each optionally substituted with OH, and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); and P.sup.9 and P.sup.10 are each independently selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, NHC.sub.1-C.sub.6alkyl each optionally substituted with OH and C.sub.1-C.sub.6alkyl, and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); with the proviso that at least one of P.sup.8, P.sup.9 or P.sup.10 is -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); P.sup.16 and each P.sup.18 are each independently selected from H, C.sub.1-C.sub.6alkyl, and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); P.sup.17 is selected from H, C.sub.1-C.sub.6alkyl, aryl, heteroaryl, C.sub.1-C.sub.6alkylaryl, C.sub.1-C.sub.6alkyl heteroaryl, C.sub.1-C.sub.6alkylaryl-Y-L-XP(O)(OR.sup.X)(OR.sup.Y) and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y), each optionally substituted with 1 to 2 substituents selected from C.sub.1-C.sub.6alkyl or heterocyclyl with the proviso that at least one of P.sup.16,P.sup.17 or a P.sup.18 contains a -Y-L-XP(O)(OR.sup.X)(OR.sup.Y) moiety; R.sup.X and R.sup.Y are independently selected from H and C.sub.1-C.sub.6alkyl; R.sup.C, R.sup.D and R.sup.H are each independently selected from H and C.sub.1-C.sub.6alkyl; X.sup.C is selected from CH and N; R.sup.E is selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, C(O)C.sub.1-C.sub.6alkyl, halogen and ((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p; X.sup.E is selected from a covalent bond, CR.sup.E2R.sup.E3 and NR.sup.E4; R.sup.E2, R.sup.E3R.sup.E4 are independently selected from H and C.sub.1-C.sub.6alkyl; X.sup.Hl-X.sup.H2 is selected from CR.sup.H2R.sup.H3, CR.sup.H2R.sup.H3-CR.sup.H2R.sup.H3, C(O)CR.sup.H2R.sup.H3, C(O)CR.sup.H2R.sup.H3, CR.sup.H2R.sup.H3C(O), NR.sup.H4C(O), C(O)NR.sup.H4, CR.sup.H2R.sup.H3S(O).sub.2 and CR.sup.H2CR.sup.H2; R.sup.H2R.sup.H3 R.sup.H4 are each independently selected from H, C.sub.1-C.sub.6alkyl and P.sup.18; X.sup.H3 is selected from N and CN; X is selected from a covalent bond, O and NH; Y is selected from a covalent bond, O, C(O), S and NH; L is selected from, a covalent bond C.sub.1-C.sub.6alkylene, C.sub.1-C.sub.6alkenylene, arylene, heteroarylene, C.sub.1-C.sub.6alkyleneoxy and ((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.peach optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, OP(O)(OH).sub.2 and P(O)(OH).sub.2; m is selected from 0 or 1; each p is independently selected from 1, 2, 3, 4, 5 and 6; q is selected from 1, 2, 3 and 4; and s is selected from 0 and 1; Formula (G) is as follows: ##STR00024## wherein: P.sup.11 is selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, NR.sup.VR.sup.W and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); P.sup.12 is selected from H, C.sub.1-C.sub.6alkyl, aryl optionally substituted by C(O)NR.sup.VR.sup.W, and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); P.sup.13, P.sup.14 and P.sup.15 are independently selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy and -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); with the proviso that at least one of P.sup.11, P.sup.12, P.sup.13, P.sup.14 P.sup.15 is -Y-L-XP(O)(OR.sup.X)(OR.sup.Y); R.sup.V and R.sup.W are independently selected from H, C.sub.1-C.sub.6alkyl or together with the nitrogen atom to which they are attached form a 4 to 7 remembered heterocyclic ring; X.sup.G is selected from C, CH and N; represents an optional double bond, wherein X.sup.G is C if is a double bond; and R.sup.G is selected from H and C.sub.1-C.sub.6alkyl; X is selected from a covalent bond, O and NH; Y is selected from a covalent bond, O, C(O), S and NH; L is selected from, a covalent bond C.sub.1-C.sub.6alkylene, C.sub.1-C.sub.6alkenylene, arylene, heteroarylene, C.sub.1-C.sub.6alkyleneoxy and ((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.peach optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, OP(O)(OH).sub.2 and P(O)(OH).sub.2; each p is independently selected from 1, 2, 3, 4, 5 and 6 and q is selected from 1, 2, 3 and 4; Formula (I) and Formula (II) are as follows: ##STR00025## wherein: Z is NH2 or OH; X.sup.1 is alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, carbocyclylene, substituted carbocyclylene, heterocyclylene, or substituted heterocyclylene; L.sup.1 is a covalent bond, arylene, substituted arylene, heterocyclylene, substituted heterocyclylene, carbocyclylene, substituted carbocyclylene, S, S(O), S(O).sub.2, or O; X.sup.2 is a covalent bond, alkylene, or substituted alkylene; L.sup.2 is NR.sup.S, N(R.sup.5)C(O) O, S, S(O), S(O).sub.2, or a covalent bond; R.sup.3 is H, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, or substituted heterocyclylalkyl; Y.sup.1 and Y.sup.2 are each independently a covalent bond, Oor -NR.sup.5-; or Y.sup.1R.sup.1 and YR.sup.2 are each independently ONC(R.sup.6R.sup.7); R.sup.1 and R.sup.2 are each independently H, alkyl, substituted alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl, substituted heterocyclylalkyl, -alkylene C(O)OR.sup.5, -(substituted alkylene)C(O)O-R.sup.5, -alkyleneOC(O)R.sup.5, -(substituted alkylene)OC(O)R.sup.5, -alkyleneOC(O)OR.sup.5, or -(substituted alkylene)OC(O)OR.sup.5 R.sup.4 is H, halogen, OH, O-alkyl, O-alkyleneOC(O)O-R.sup.5, OC(O)OR.sup.5, -SH, or -NH(R.sup.5); each R.sup.5, R.sup.6, and R.sup.7 are independently H, alkyl, substituted alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl, or substituted heterocyclylalkyl; Formula (J) is as follows: ##STR00026## wherein: R.sup.1 is H, C(O) -C.sub.7-C.sub.18alkyl or C(O)C.sub.1-C.sub.6alkyl; R.sup.2 is C.sub.7-C.sub.18alkyl ; R.sup.3 is C.sub.7-C.sub.18alkyl; L.sub.1 is -CH.sub.2OC(O), -CH.sub.2O, -CH.sub.2NR.sup.7C(O) or -CH.sub.2OC(O)NR.sup.7-; L.sub.2 is -OC(O), O, -NR.sup.7C(O) or -OC(O)NR.sup.7-; R.sup.4 is L.sub.3R.sup.5 or L.sub.4R.sup.5; R.sup.5 is N(R.sup.7).sub.2, -OR.sup.7, P(O)(OR.sup.7).sub.2, C(O)OR.sup.7, -NR.sup.7C(O)L.sub.3R.sup.8, -NR.sup.7C(O)L.sub.4R.sup.8, -OL.sub.3R.sup.6, C (O)NR.sup.7L.sub.3R.sup.8, C(O)NR.sup.7L.sub.4R.sup.8, S(O).sub.2OR.sup.7, -OS(O).sub.2OR.sup.7, C.sub.1-C.sub.6alkyl, a C.sub.6aryl, a C.sub.10aryl, a C.sub.14aryl, 5 to 14 ring membered heteroaryl containing 1 to 3 heteroatoms selected from O, S and N, C.sub.3-C.sub.8cycloalkyl or a 5 to 6 ring membered heterocycloalkyl containing 1 to 3 heteroatoms selected from O, S and N, wherein the aryl, heteroaryl, cycloalkyl and heterocycloalkyl of R.sup.5 are each unsubstituted or the aryl, heteroaryl, cycloalkyl and heterocycloalkyl of R.sup.5 are each substituted with 1 to 3 substituents independently selected from -OR.sup.9, -OL.sub.3R.sup.6, -OL.sub.4R.sup.6, -OR.sup.7, and C(O)OR.sup.7; L.sub.3 is a Ci-Cioalkylene, wherein the Ci-Cioalkylene of L.sub.3 is unsubstituted, or the C.sub.1-C.sub.10alkylene of L.sub.3 is substituted with 1 to 4 R.sup.6 groups, or the C.sub.1-C.sub.10alkylene of L.sub.3 is substituted with 2 C.sub.1-C.sub.6alkyl groups on the same carbon atom which together, along with the carbon atom they are attached to, form a C.sub.3-C.sub.8cycloakyl; L.sub.4 is ((CR.sup.7R.sup.7).sub.pO).sub.q(CR.sup.10R.sup.10).sub.p or (CR.sup.11R.sup.11)((CR.sup.7R.sup.7).sub.pO).sub.q(CR.sup.10R.sup.10).sub.p, wherein each R.sup.11 is a C.sub.1-C.sub.6alkyl groups which together, along with the carbon atom they are attached to, form a C.sub.3-C.sub.8cycloakyl; each R.sup.6 is independently selected from halo, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkyl substituted with 1-2 hydroxyl groups, -OR.sup.7, N(R.sup.7).sub.2, C(O)OH, C(O)N(R.sup.7).sub.2, P(O)(OR.sup.7).sub.2, a C.sub.6aryl, a C.sub.10aryl and a C.sub.14aryl; each R.sup.7 is independently selected from H and C.sub.1-C.sub.6alkyl; R.sup.8 is selected from SR.sup.7, C(O)OH, P(O)(OR.sup.7).sub.2, and a 5 to 6 ring membered heterocycloalkyl containing 1 to 3 heteroatoms selected from O and N; R.sup.9 is phenyl; each R.sup.10 is independently selected from H and halo; each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or 4; Formula (K) is as follows: ##STR00027## wherein: R.sup.1 is H, C.sub.1-C.sub.6alkyl, C(R.sup.5).sub.2OH, L.sup.1R.sup.5, L.sup.1R.sup.6, L.sup.2R.sup.5, L.sup.2R.sup.6, OL.sup.2R.sup.5, or -OL.sup.2R.sup.6; L.sup.1 is C(O) or 0; L.sup.2 is C.sub.1-C.sub.6alkylene, C.sub.2-C.sub.6alkenylene, arylene, heteroarylene or ((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p, wherein the C.sub.1-C.sub.6alkylene and C.sub.2-C.sub.6alkenylene of L.sup.2 are optionally substituted with 1 to 4 fluoro groups; each L.sup.3 is independently selected from C.sub.1-C.sub.6alkylene and ((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p, wherein the C.sub.1-C.sub.6alkylene of L.sup.3 is optionally substituted with 1 to 4 fluoro groups; L.sup.4 is arylene or heteroarylene; R.sup.2 is H or C.sub.1-C.sub.6alkyl; R.sup.3 is selected from C.sub.1-C.sub.4alkyl, L.sup.3R.sup.5, L.sup.1R.sup.5, L.sup.3R.sup.7, L.sup.3L.sup.4L.sup.3R.sup.7, L.sup.3L.sup.4R.sup.5, L.sup.3L.sup.4L.sup.3R.sup.5, OL.sup.3R.sup.5, OL.sup.3R.sup.7, OL.sup.3L.sup.4R.sup.7, OL.sup.3L.sup.4L.sup.3R.sup.7, OL.sup.3L.sup.4R.sup.5, OL.sup.3L.sup.4L.sup.3R.sup.5 and C(R.sup.5).sub.2OH ; each R.sup.4 is independently selected from H and fluoro; R.sup.5 is P(O)(OR.sup.9).sub.2, R.sup.6 is CF.sub.2P(O)(OR.sup.9).sub.2 or C(O)OR.sup.10; R.sup.7 is CF.sub.2P(O)(OR.sup.9).sub.2 or C(O)OR.sup.10; R.sup.8 is H or C.sub.1-C.sub.4alkyl; each R.sup.9 is independently selected from H and C.sub.1-C.sub.6alkyl; R.sub.10 is H or C.sub.1-C.sub.4alkyl; each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or 4; Formula (F) is as follows: ##STR00028## wherein: X.sup.3 is N; X.sup.4 is N or CR.sup.3 X.sup.5 is CR.sup.4CR.sup.5; R.sup.1 and R.sup.2 are H; R.sup.3 is H; R.sup.4 and R.sup.5 are each independently selected from H, halogen, C(O)OR.sup.7, C(O)R.sup.7, C(O)N(R.sup.11.sub.R.sup.12).sub., N(R.sup.11R.sup.12), N(R.sup.9).sub.2, NHN(R.sup.9).sub.2, SR.sup.7, -(CH.sub.2).sub.nOR.sup.7, -(CH.sub.2).sub.nR.sup.7, LR.sup.8, LR.sup.10, OLR.sup.8, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, and C.sub.3-C.sub.8heterocycloalkyl, wherein the C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, and C.sub.3-C.sub.8heterocycloalkyl groups of R.sup.4 and R.sup.5 are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, -NO.sub.2, -R.sup.7, -OR.sup.8, C(O)R.sup.8, -OC(O)R.sup.8, C(O)OR.sup.8, -N(R.sup.9).sub.2, P(O)(OR.sup.8).sub.2, -OP(O)(OR.sup.8).sub.2,) P(O)(OR.sup.10).sub.2. -OP(O)(OR.sup.10).sub.2, C(O)N(R.sup.9).sub.2, S(O).sub.2R.sup.8, S(O)R.sup.8, S(O).sub.2N(R.sup.9).sub.2, and -NR.sup.9S(O).sub.2R.sup.8; or, R.sup.3 and R.sup.4, or R.sup.4 and R.sup.5, or R.sup.5 and R.sup.6, when present on adjacent ring atoms, can optionally be linked together to form a 5-6 membered ring, wherein the 5-6 membered ring is optionally substituted with R.sup.7; each L is independently selected from a bond, (O(CH.sub.2).sub.m).sub.t, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenylene and C.sub.2-C.sub.6alkynylene, wherein the C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenylene and C.sub.2-C.sub.6alkynylene of L are each optionally substituted with 1 to 4 substituents independently selected from halogen, -R.sup.8, -OR.sup.8, N(R.sup.9).sub.2, P(O)(OR.sup.8).sub.2, -OP(O)(OR.sup.8).sub.2, P(O)(OR.sup.10).sub.2, and -OP(O)(OR.sup.10).sub.2; R.sup.7 is selected from H, C.sub.1-C.sub.6alkyl, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy, and C.sub.3-C.sub.8heterocycloalkyl, wherein the C.sub.1-C.sub.6alkyl, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy, and C.sub.3-C.sub.8heterocycloalkyl groups of R.sup.7 are each optionally substituted with 1 to 3 R.sup.13 groups, and each R.sup.13 is independently selected from halogen, -CN, LR.sup.9, -LOR.sup.9, -OLR.sup.9, -LR.sup.10, -LOR.sup.10, -OLR.sup.10, LR.sup.8, -LOR.sup.8, OLR.sup.8, -LSR.sup.8, -LSR.sup.10, -LC(O)R.sup.8, -OLC(O)R.sup.8, -LC(O).sup.8, -LC(O)R.sup.10, -LOC (O)OR.sup.8, -LC(O)NR.sup.9R.sup.11, -LC(O)NR.sup.9R.sup.8, -LN(R.sup.9).sub.2, -LNR.sup.9R.sup.8, -LNR.sup.9R.sup.1 , -LC(O)N(R.sup.9).sub.2, -LS (O).sub.2R.sup.8, -LS(O)R.sup.8, -LC(O)NR.sup.8OH, -LNR.sup.9C(O)R.sup.8, -LNR.sup.9C(O)OR.sub.2, -LS(O).sub.2N(R.sup.9).sub.2, -OLS (O).sub.2N(R.sup.9).sub.2, -LNR.sup.9S(O).sub.2R.sup.8, -LC(O)NR.sup.9LN(R.sup.9).sub.2, -LP(O)(OR.sup.8).sub.2, -LOP(O)(OR.sup.8).sub.2, -LP (O)(OR.sup.10).sub.2 and -OLP(O)(OR.sup.10; each R.sup.8 is independently selected from H, -CH(R.sup.10).sub.2, C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkene, C.sub.2-c.sub.8alkyne, C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6heteroalkyl, C.sub.3-C.sub.8cycloalkyl, C.sub.2-C.sub.8heterocycloalkyl, C.sub.1-C.sub.6hydroxyalkyl and C.sub.1-C.sub.6haloalkoxy, wherein the C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy, C.sub.3-C.sub.8cycloalkyl, C.sub.2-C.sub.8heterocycloalkyl, C.sub.1-C.sub.6hydroxyalkyl and C.sub.1-C.sub.6haloalkoxy groups of R.sup.8 are each optionally substituted with 1 to 3 substituents independently selected from -CN, R.sup.11, -OR.sup.11, -SR.sup.11, -C (O)R.sup.11, -OC(O)R.sup.11, C(O)N(R.sup.9).sub.2, C(O)OR.sup.11, -NR.sup.9C(O)R.sup.11, -NR.sup.9R.sup.10, -NR.sup.11R.sup.12, -N(R.sup.9).sub.2, OR.sup.9, -OR.sup.10, C(O)NR.sup.11R.sup.12, C(O)NR.sup.11OH, S(O).sub.2R.sup.11, S(O)R.sup.11, S(O.sub.2NR.sup.11R.sup.12, -NR.sup.11S(O.sub.2R.sup.11, P(O)(OR.sup.11).sub.2, and -OP(O)(OR.sup.11).sub.2; each R.sup.9 is independently selected from H, C(O)R.sup.8, C(O)OR.sup.8, C(O)R.sup.10, C(O)OR.sup.10, S(O).sub.2R.sup.10, -C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl and C.sub.3-C.sub.6 cycloalkyl, or each R.sup.9 is independently a C.sub.1-C.sub.6alkyl that together with N they are attached to form a C.sub.3-C.sub.8heterocycloalkyl, wherein the C.sub.3-C.sub.8heterocycloalkyl ring optionally contains an additional heteroatom selected from N, O and S, and wherein the C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, C.sub.3-C.sub.6 cycloalkyl, or C.sub.3-C.sub.8heterocycloalkyl groups of R.sup.9 are each optionally substituted with 1 to 3 substituents independently selected from -CN, R.sup.11, -OR.sup.11, -SR.sup.11, C(O)R.sup.11, OC(O)R.sup.11, C(O)OR.sup.11, -NR.sup.11R.sup.12, C(O)NR.sup.11R.sup.12, C(O)NR.sup.11OH, S(O)2R.sup.11, S(O)R.sup.11, S(O).sub.2NR.sup.11R.sup.12, -NR.sup.11S(O).sub.2R.sup.11, P(O)(OR.sup.11).sub.2 and -OP (O)(OR.sup.11).sub.2; each R.sup.10 is independently selected from aryl, C.sub.3-C.sub.8cycloalkyl, C.sub.3-C.sub.8heterocycloalkyl and heteroaryl, wherein the aryl, C.sub.3-C.sub.8cycloalkyl, C.sub.3-C.sub.8heterocycloalkyl and heteroaryl groups are optionally substituted with 1 to 3 substituents selected from halogen, R.sup.8, -OR.sup.8, -LR.sup.9, -LOR.sup.9, N(R.sup.9).sub.2, NR.sup.9C(O)R.sup.8, NR.sup.9CO.sub.2R.sup.8. C(O).sub.2R.sup.8, C(O)R.sup.8 and C(O)N(R.sup.9).sub.2; R.sup.11 and R.sup.12 are independently selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6aloalkyl, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, and C.sub.3-C.sub.8heterocycloalkyl, wherein the C.sub.1-C.sub.6aIkyl, C .sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, and C.sub.3-C.sub.8heterocycloalkyl groups of R.sup.11 and R.sup.12 are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, R.sup.8, -OR.sup.8, C(O)R.sup.8, OC(O)R.sup.8, C(O)OR.sup.8, N(R.sup.9).sub.2, -NR .sup.8C(O)R.sup.8, -NR.sup.8C(O)OR.sup.8, C(O)N(R.sup.9).sub.2, C.sub.3-C.sub.8heterocycloalkyl, S(O).sub.2, S(O).sub.2N(R.sup.9).sub.2, -NR.sup.9S(O).sub.2R.sup.8, C.sub.1-C.sub.6haloalkyl and C.sub.1-C.sub.6haloalkoxy; or R.sup.11 and R.sup.12 are each independently C.sub.1-C.sub.6alkyl and taken together with the N atom to which they are attached form an optionally substituted C.sub.3-C.sub.8heterocycloalkyl ring optionally containing an additional heteroatom selected from N, O and S; ring A is an aryl or a heteroaryl, wherein the aryl and heteroaryl groups of Ring A are optionally substituted with 1 to 3 R.sup.A groups, wherein each R.sup.A is independently selected from -R.sup.8, -R7, -OR.sup.7, -OR.sup.8, -R.sup.10, -OR.sup.10, SR.sup.8, NO.sub.2, -CN, N(R.sup.9).sub.2, -NR.sup.9C(O)R.sup.8, -NR.sup.9C(S)R.sup.8, -NR.sup.9C(O)N(R.sup.9).sub.2, - NR.sup.9C(S)N(R.sup.9).sub.2, -NR.sup.9CO.sub.2R.sup.8, -NR.sup.9NR.sup.9C(O)R.sup.8, -NR.sup.9NR.sup.9C(O)N(R.sup.9).sub.2, -NR.sup.9NR.sup.9CO.sub.2R.sup.8, C(O)C(O)R.sup.8, C(O)CH.sub.2C(O)R.sup.8, -CO.sub.2R.sup.8, -(CH.sub.2).sub.nCO.sub.2R.sup.8, C(O)R.sup.8, C(S)R.sup.8, C(O)N(R.sup.9).sub.2, C(S)N(R.sup.9).sub.2, -OC(O)N(R.sup.9).sub.2, -OC(O)R.sup.8, C(O)N(OR.sup.8)R.sup.8, C(NOR.sup.8)R.sup.8, S(O).sub.2R.sup.8, S(O).sub.3R.sup.8, -SO.sub.2N(R.sup.9).sub.2, S(O)R.sup.8, -NR.sup.9SO.sub.2N(R.sup.9).sub.2, -NR.sup.9SO.sub.2R.sup.8, P(O)(OR.sup.8).sub.2, -OP(O)(OR.sup.8).sub.2, P(O)(OR.sup.10).sub.2, -OP(O)(OR.sup.10).sub.2, N(OR.sup.8)R.sup.8, -CHCHCO.sub.2R.sup.8, C(NH)N(R.sup.9).sub.2, and -(CH.sub.2).sub.nNHC(O)R.sup.8 or two adjacent R.sup.A substituents on Ring A form a 5-6 membered ring that contains up to two heteroatoms as ring members; n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7 or 8; each m is independently selected from 1, 2, 3, 4, 5 and 6; and t is 1, 2, 3, 4, 5, 6, 7 or 8.

12. The immunogenic composition of claim 1, further comprising a histidine buffer.

13. The immunogenic composition of claim 1, with a diphtheria toxoid concentration 4 Lf/ml.

14. The immunogenic composition of claim 1, with a tetanus toxoid concentration 9 Lf/ml.

15. The immunogenic composition of claim 1, with a pertussis toxoid concentration 4 g/ml.

16. The immunogenic composition of claim 1, further comprising a trivalent inactivated poliovirus antigen component.

17. The immunogenic composition of claim 1, having a pH between 6.1 and 7.9.

18. A method of raising an immune response in a subject, comprising the step of administering to the subject the composition of claim 1.

19. The method of claim 18, wherein the subject has previously received a DTP vaccine as a child.

20. An immunogenic composition comprising a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, and an oil-in-water emulsion adjuvant, wherein the immunogenic composition comprises an excess (measured in Lf units) of tetanus toxoid relative to diphtheria toxoid.

21. The immunogenic composition of claim 20, wherein the emulsion comprises squalene and/or polysorbate 80.

22. The immunogenic composition of claim 20, wherein at least 80% by number of oil droplets in the emulsion have a diameter of less than 220 nm.

23. The immunogenic composition of claim 20, with a diphtheria toxoid concentration 4 Lf/ml.

24. The immunogenic composition of claim 20, with a tetanus toxoid concentration 9 Lf/ml.

25. The immunogenic composition of claim 20, with a pertussis toxoid concentration 4 g/ml.

26. The immunogenic composition of claim 20, further comprising a trivalent inactivated poliovirus antigen component.

27. The immunogenic composition of claim 20, having a pH between 6.1 and 7.9.

28. A method of raising an immune response in a subject, comprising the step of administering to the subject the immunogenic composition of claim 21.

29. The method of claim 28, wherein the subject has previously received a DTP vaccine as a child.

30. An immunogenic composition comprising an acellular pertussis component in which a pertussis toxoid, filamentous hemagglutinin and pertactin are present at a mass ratio of 1:1:2, wherein the pertussis toxoid is the PT-9K/129G double mutant.

31. The immunogenic composition of claim 30, in unit dose form, and comprising 4 g pertussis toxoid, 4 g FHA and 8 g pertactin per unit dose.

32. An immunogenic composition comprising an acellular pertussis component in which a pertussis toxoid, filamentous hemagglutinin and pertactin are present at a mass ratio of 1:1:2, the immunogenic composition further comprising diphtheria toxoid and tetanus toxoid.

33. An immunogenic composition comprising an acellular pertussis component in which a pertussis toxoid, filamentous hemagglutinin and pertactin are present at a mass ratio of 1:1:2, the immunogenic composition further comprising a TLR agonist adsorbed to an aluminium salt adjuvant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the % of FHA-specific memory B cells for the indicated treatment groups.

MODES FOR CARRYING OUT THE INVENTION

(2) Vaccine Preparation

(3) References 31 and 83 disclose TLR7 agonists having formula (K) as discussed above. One of these compounds, 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo [f]-[1,7]naphthyridin-8-yl)propanoic acid is referred to hereafter as compound K2:

(4) ##STR00020##

(5) Compound K2 is added to water at 4mg/ml, then 1M NaOH is added to ensure full solubilisation, with stirring for 15 minutes at room temperature. This material is added to a suspension of aluminium hydroxide adjuvant (AlH) to give the desired final concentration. This mixture is shaken for 2 hours at ambient temperature to ensure full adsorption, and then histidine buffer components are added (10mM histidine buffer, pH 6.5).

(6) The compound can also be used as an arginine salt monohydrate (obtained by mixing 98 mg of the compound with 1.7 ml of 0.1 M arginine in 80/20 methanol/water to give a 57 mg/ml solution, followed by addition of 7 ml ethanol to precipitate the salt) in which case it is seen that the NaOH is not required for solubilisation prior to mixing with the AlH.

(7) Four different mixtures are prepared, giving a final K2 concentration of 10, 50, 250 or 500 g/ml (to provide a 1, 5, 25 or 50 g dose of K2 in a 100 l dosage volume); the AlH concentration is always 3 mg/ml. At all strengths >95% of compound K2 is adsorbed to the AlH. The adsorbed adjuvant is referred to hereafter as AlH/K2.

(8) Adjuvant Adsorption to Antigens

(9) 3-valent (DTaP) vaccines were adjuvanted with AlH alone or with AlH/K2. These formulations showed optimal pH (6.5-6.80.1) and osmolarity values (0.30050 mO). Osmolarity was adjusted with NaCl. Adsorption was detected by separating the adjuvant-antigen complexes from unadsorbed antigens by centrifugation. 0.4% DOC was added to the supernatant containing the unadsorbed antigens. Antigens were precipitated by the addition of 60% TCA and collected by centrifugation. The pellet containing the TCA-precipitated antigens was resuspended in loading buffer and loaded onto an SDS-PAGE gel. The pellet containing the adjuvant-antigen complexes was resuspended in desorption buffer (4 concentration: 0.5 M Na2HPO.sub.4 pH, 8 g SDS, 25 g glycerol, 6.16 g DTT and bromophenol blue), the aluminium hydroxide was removed by centrifugation and the supernatant applied to an SDS-PAGE gel.

(10) Using AlH alone at a concentration of 2 mg/ml, the adsorption profiles for DT, TT, PT, FHA and 69K detected by Coomassie Blue staining were complete. No bands were detected in the DOC-TCA-treated supernatants. Western Blot analysis confirmed complete AlH adsorption for DT, TT, PT, FHA and 69K.

(11) Four different K2 concentrations were tested (0.1, 0.025, 0.01, 0.005 mg/ml). The AlH concentration was constant at 2 mg/ml. Even at 0.1 mg/ml K2 all antigens were completely adsorbed.

(12) Immunogenicity Testing with TLR Agonists

(13) Four vaccines were tested, each containing (per 0.5 ml) 5 Lf tetanus toxoid, 2 Lf of diphtheria toxoid, and 16 g acellular pertussis antigens (a mixture of purified PT-9K/129G, FHA and p69 pertactin).

(14) The four vaccines were (A) unadjuvanted (B) adjuvanted with 2 mg/ml AlH (C) adjuvanted with 2 mg/ml AlH plus 100 g/ml synthetic monophosphoryl lipid A i.e. a TLR4 agonist, or (D) adjuvanted with 2 mg/ml AlH plus 1 mg/ml compound K2 i.e. a TLR7 agonist. The TLR agonists in vaccines (C) and (D) were adsorbed to the AlH. All antigens were adsorbed to the AlH in formulations (B), (C) and (D).

(15) For comparison the BOOSTRIX product was also tested. As discussed above it contains (per 0.5 ml) 2.5 Lf of diphtheria toxoid, 5 Lf tetanus toxoid, and 18.5 g acellular pertussis antigens (a mixture of purified PT, FHA and p69 pertactin), and it is adjuvanted with a mixture of aluminium phosphate and hydroxide salts. A mixture of buffer and AlH was used as a negative control.

(16) The four vaccines were administered to female Balb/C mice (6 weeks old) at 100 l intramuscular doses on days 0, 21 and 35. Sera were tested 2 weeks after each dose.

(17) Serum total IgG titers were measured against each antigen and were as follows (geometric means):

(18) TABLE-US-00002 Day Ag Unadj AlH AlH + K2 AlH + MPL Boostrix ve control 14 Dt 0.030 0.603 8.119 1.762 1.205 0.030 Tt 0.191 2.546 46.14 14.49 3.217 0.030 PT 14.28 10.26 26.68 16.61 4.685 1.080 FHA 0.145 0.579 27.38 7.942 1.458 0.126 p69 0.566 7.046 72.47 38.49 12.39 0.129 35 Dt 0.030 69.29 490.1 139.3 89.59 0.030 Tt 48.98 128.6 808.9 298.0 109.8 0.030 PT 245.0 267.5 377.1 695.9 195.0 2.734 FHA 7.847 65.25 400.6 231.0 49.49 0.030 p69 32.50 222.2 1484 575.8 318.0 0.050 49 Dt 0.055 79.74 452.9 149.2 88.95 0.047 Tt 60.59 123.2 694.3 317.0 105.6 0.030 PT 200.8 387.7 329.7 642.3 300.0 2.065 FHA 20.96 72.10 462.4 272.5 78.98 0.030 p69 107.6 384.9 1275 794.5 302.6 0.134

(19) Thus in all cases and at all time points (except for PT at day 49) the highest titers in these 6 groups were seen in the mice which had received the antigens adjuvanted with adsorbed TLR agonist, and the addition of a TLR agonist to AlH improved IgG responses relative to AlH alone. Importantly, improved responses were seen in all cases when compared to the licensed BOOSTRIX vaccine. Moreover, unlike AlH alone or BOOSTRIX, the adsorbed TLR agonists were consistently able to improve anti-PT titers relative to the unadjuvanted group.

(20) The use of the TLR agonists also leads to more rapid responses. The second dose showed a clear increase in IgG responses for all antigens, but the improvements after the third dose were not so significant. The mice in these experiments were naive to DTP but in a real-world human situation the target patients will previously have received DTP vaccine as a child and so the rapid response seen after the second dose in these experiments is helpful.

(21) Immunogenicity Testing with Oil-in-Water Emulsion

(22) Vaccines were prepared containing (per 0.5 ml) 5 Lf tetanus toxoid, 2 Lf of diphtheria toxoid, and 16 g acellular pertussis antigens (a mixture of purified PT-9K/129G, FHA and p69 pertactin). These were administered to female Balb/C mice (6 weeks old) at 100 l intramuscular doses on days 0, 21 and 35. Sera were tested 2 weeks after each dose. Vaccines were (A) unadjuvanted or (B) adjuvanted with MF59 emulsion, by mixing 50 l antigen solution with 50 l MF59. For comparison the BOOSTRIX product was also tested, and an antigen-free negative control was also tested.

(23) Serum total IgG titers were measured against each antigen and were as follows (geometric means):

(24) TABLE-US-00003 Day Ag Unadj MF59 Boostrix ve control 14 Dt 0.030 0.038 1.308 0.030 Tt 0.064 3.025 2.480 0.030 PT 7.255 9.448 1.629 1.517 FHA 0.034 0.026 0.844 0.137 p69 1.365 9.281 14.46 1.670 35 Dt 0.030 115.2 85.55 0.033 Tt 24.31 379.6 83.61 0.060 PT 247.4 391.9 112.7 3.582 FHA 7.433 148.8 37.58 0.068 p69 11.44 1006 398.3 3.817 49 Dt 0.5165 78.54 92.18 0.054 Tt 40.21 337.5 99.09 0.030 PT 353.6 480.8 162.8 5.231 FHA 13.32 218.1 65.34 0.119 p69 36.91 1036 403.6 1.952

(25) Thus after two doses in DTP-naive mice the emulsion-adjuvanted vaccine gave much better antibody titers than the approved BOOSTRIX product. This superiority was maintained after a third dose, except for the anti-Dt response. Moreover, unlike BOOSTRIX, the emulsion was able to improve anti-PT titers relative to the unadjuvanted group.

(26) FHA-Specific Memory B Cells

(27) Four-to-five months after the third dose, FHA-specific memory B cells were measured in the immunised mice. The mice were sacrificed and their spleen cells were cultured in the presence of IL-2 and CpG for 5 days in order to expand all memory B cells. Spleen cells were then harvested and seeded in 96-well ELISPOT plates previously coated with either FHA antigen (10 mg/ml) or anti-mouse Ig. After overnight incubation, plates were washed to remove unattached spleen cells and both FHA-specific and total memory B cells were detected by biotinylated anti-mouse Ig and HRP-streptavidin. Colored spots, representing individual memory B cell, were counted with an ELISPOT reader instrument. The percentage of FHA-specific B cells compared to total B cells was then calculated for each sample, and FIG. 1 shows the results for the following groups: (A) unadjuvanted; (B) adjuvanted with AlH; (C) adjuvanted with AlH plus MPL-A; (D) adjuvanted with AlH plus K2; (E) adjuvanted with MF59; (F) BOOSTRIX; and (G) negative control. The highest response was seen in group (D) i.e. using the adsorbed TLR7 agonist. The next highest responses were seen in groups (C) and (E), with either the adsorbed TLR4 agonist or the emulsion, where responses were essentially the same but were still higher than with the BOOSTRIX product.

(28) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

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