Combination vaccines with lower doses of antigen and/or adjuvant

Abstract

Combination vaccine compositions as well as methods for their manufacture have a relatively low amount of antigen and/or a relatively low amount of aluminium, but they can nevertheless have immunogenicity which is comparable to combination vaccines with a relatively high amount of antigen and/or a relatively high amount of aluminium. Aluminium-free combination vaccine compositions are also provided e.g. compositions which are adjuvanted with an oil-in-water emulsion adjuvant.

Claims

1. An immunogenic composition in a unit dose form for administration to a patient comprising (i) a diphtheria toxoid, a tetanus toxoid, and an acellular pertussis antigen which contains a pertussis toxoid, (ii) an aluminium salt adjuvant, wherein the amount of Al.sup.+++ in the unit dose is less than 0.2 mg, and (iii) a toll-like receptor 7 (TLR 7) agonist, wherein at least 50% (by weight) of the TLR 7 agonist is adsorbed to the aluminium salt adjuvant.

2. The composition of claim 1, further comprising one or more of (i) a Hib conjugate, (ii) a hepatitis B virus surface antigen, and/or (iii) an inactivated poliovirus antigen.

3. An immunogenic composition in a unit dose form for administration to a patient comprising (i) a low dose of each of a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, (ii) an aluminium salt adjuvant, wherein the amount of Al.sup.+++ in the unit dose is less than 0.2 mg, and (iii) a TLR 7 agonist, wherein at least 50% (by weight) of the TLR 7 agonist is adsorbed to the aluminium salt adjuvant.

4. The composition of claim 3, wherein the composition has 8 Lf/ml diphtheria toxoid.

5. The composition of claim 3, wherein the composition has 3.5 Lf/ml tetanus toxoid.

6. The composition of claim 3, wherein the acellular pertussis antigen has 5 g/ml pertussis toxoid.

7. The composition of claim 3, further comprising a low dose of an H. influenzae type b (Hib) conjugate.

8. The composition of claim 7, wherein the composition has 5 g/ml Hib saccharide.

9. The composition of claim 3, further comprising a low dose of surface antigen of the hepatitis B virus (HBsAg).

10. The composition of claim 9, wherein the composition has 5 g/ml HBsAg.

11. The composition of claim 3, further comprising a low dose of inactivated poliovirus.

12. The composition of claim 11, wherein the composition has (i) 20 DU/ml type 1 poliovirus and/or (ii) 4 DU/ml type 2 poliovirus and/or (iii) 16 DU/ml type 3 poliovirus.

13. The composition of claim 3, wherein the aluminium salt adjuvant is (i) an aluminium hydroxide adjuvant or (ii) an aluminium phosphate adjuvant or (iii) a mixture of an aluminium hydroxide adjuvant and an aluminium phosphate adjuvant.

14. The composition of claim 3, wherein the aluminium salt adjuvant is an aluminium hydroxide adjuvant.

15. The composition of claim 1, wherein the at least 50% (by weight) of the TLR agonist adsorbed to the aluminium salt adjuvant is in a stable complex with the aluminium salt adjuvant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) There are no drawings.

MODES FOR CARRYING OUT THE INVENTION

(2) Adjuvant Adsorption to Antigens

(3) 3-valent (DTaP) or 6-valent (DTaP-HBsAg-IPV-Hib) vaccines were adjuvanted with aluminium hydroxide alone, aluminium hydroxide with pre-adsorbed compound T, poly(lactide-co-glycolide) microparticles (PLG), and MF59 oil-in-water emulsion. Aluminium hydroxide and aluminium hydroxide with pre-adsorbed compound T were prepared in histidine buffer pH 6.5. At pH 6.5, aluminium hydroxide has a positive net charge, while most proteins have a negative net charge. The pH value was chosen to provide good adsorption of most of the tested antigens. All formulations adjuvanted with aluminium hydroxide or aluminium hydroxide with pre-adsorbed compound T showed optimal pH (6.5-6.80.1) and osmolarity values (0.30050 mO). Osmolarity was adjusted with NaCl. Antigens for the MF59-adjuvanted formulations were prepared in PBS. The resulting preparations had pH values between 6.2 and 7.3 and osmolarity values around 0.30050 mO. Formulations containing PLG microparticles were prepared in water. PLG formulations showed suboptimal osmolarity values. The pH of the PLG formulations ranged from 5.8 to 6.50.1. The PLG microparticles were prepared with dioctylsulfosuccinate (DSS) which confers a negative net charge to the microparticles. Thus interaction of the microparticles with the antigen is mediated by positive charges on the antigen surface.

(4) For aluminium hydroxide alone, aluminium hydroxide with pre-adsorbed compound T, and PLG, 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 Na.sub.2HPO.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. The MF59 oil-in-water emulsion containing antigens were separated by centrifugation in an oily phase and an aqueous phase. Both the aqueous phase containing unabsorbed antigens and the oily phase presumably containing MF59-associated antigens were mixed with loading buffer and applied to an SDS-PAGE gel. After electrophoretic separation of the samples, the gels were either analysed by Coomassie Blue staining or by Western blotting.

(5) Using aluminium hydroxide 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 both for the 3-valent formulation and the 6-valent formulation. No bands were detected in the DOC-TCA-treated supernatants. Western Blot analysis confirmed complete aluminium hydroxide adsorption for DT, TT, PT, FHA and 69K for both the 3-valent formulation and the 6-valent formulation. Likewise, the other five antigensIPV1, IPV2, IPV3, HBsAg and Hib-CRMdid not show any detectable bands in the DOC-TCA-treated supernatants of aluminium hydroxide-adsorbed formulations. Thus all ten antigens present in the 6-valent formulation completely adsorbed to aluminium hydroxide.

(6) For aluminium hydroxide with pre-adsorbed compound T, antigen adsorption differed between the 3-valent formulation and the 6-valent formulation. Four different compound T concentrations were tested (0.1, 0.025, 0.01, 0.005 mg/ml). The aluminium hydroxide concentration was kept constant at 2 mg/ml. At 0.1 mg/ml compound T, all antigens in the 3-valent formulation were completely adsorbed. In contrast, 69K and PT in the 6-valent formulation were not completely adsorbed as determined by Coomassie Blue staining. At 0.01 mg/ml compound T, Western blot analysis confirmed adsorption of all ten antigens in the 6-valent formulation. Only a small amount of TT was still detectable in the supernatant using Western blot. The fact that TT could be detected in the supernatant by Western blot but not by SDS-PAGE is likely due to the greater sensitivity of the former method. Thus, at higher concentrations, compound T appears to compete with the antigens for binding to the adjuvant. This could explain why the effect only becomes apparent in the presence of a greater number of antigens, i.e., when less aluminium hydroxide per antigen is available.

(7) Using PLG microparticles, DT, TT, IPV1, IPV2, IPV3, FHA and CRM of the Hib-CRM conjugate were mostly presented on the supernatants with only very small amounts of DT, IPV1, IPV2 and FHA being detected by Western blot in the pellet containing the antigen-adjuvant complexes. 69K and PT seemed to be presented in similar amounts in supernatant and pellet. HBsAg could neither be detected in the supernatant nor in the pellet of the PLG formulations. In comparison to preparations containing aluminium hydroxide or aluminium hydroxide with pre-adsorbed compound T, PLG absorbed significantly less antigen. Moreover, the antigen adsorption profiles obtained using PLG showed an opposite trend to those seen in the presence of the other two adjuvants probably reflecting the negative net charge of PLG versus the positive net charge of aluminium hydroxide or aluminium hydroxide with pre-adsorbed compound T.

(8) MF59 is a delivery system generally considered unable to physically interact with the antigens as shown by the lack of an antigen deposition at the injection site and independent clearance of MF59 and the antigens (see references 124 and 125). 1:1, 1:3 and 1:10 ratios (v:v of MF59 to complete antigen formulation) were tested. For all three tested ratios, SDS-PAGE and Western blot analysis showed that all ten tested antigens were present in the aqueous phase of MF59-adjuvanted formulations. Thus the antigen profiles of MF59-adjuvanted formulations corresponded to the profiles of unadjuvanted formulations. The results confirmed that MF59 does not interact with any of the tested antigens.

(9) Replacement or Reduction of Aluminium Salt Adjuvants

(10) The INFANRIX HEXA product from GlaxoSmithKline contains 30 IU diphtheria toxoid, 40 IU tetanus toxoid, an acellular pertussis component (25/25/8 g of PT/FHA/pertactin), 10 g HBsAg, a trivalent IPV component (40/8/32 DU of types 1/2/3), and 10 g Hib conjugate. The vaccine is presented as a 5-valent aqueous vaccine which is used to reconstitute the Hib conjugate from its lyophilised form, to give a 0.5 ml aqueous unit dose for human infants which contains 0.95 mg aluminium hydroxide and 1.45 mg aluminium phosphate.

(11) To investigate alternative adjuvants (see above) a 6-valent mixture was adjuvanted with aluminium hydroxide alone (2 mg/ml in histidine buffer), with aluminium hydroxide with pre-adsorbed compound T (see above; 1 mg/ml), with poly(lactide-co-glycolide) microparticles (PLG, used at 40 mg/ml), or with the MF59 oil-in-water emulsion (mixed at equal volume with antigens in phosphate-buffered saline). The same diluents were used in all mouse experiments described below. Osmolarity of the formulations was adjusted with NaCl where necessary. An adjuvant-free control was also prepared. Antigen concentrations were as follows (per ml):

(12) TABLE-US-00002 DT TT PT FHA Pertactin 36.9 Lf 14.8 Lf 36.9 g 36.9 g 11.8 g IPV Type 1 IPV Type 2 IPV Type 3 HBsAg Hib 59.1 DU 11.8 DU 47.3 DU 14.8 g 14.8 g

(13) The same adjuvants were also used with a 3-valent D-T-Pa mixture (same concentrations).

(14) Osmolarity and pH were measured (and, if necessary, adjusted) after combining the components in order to ensure physiological acceptability. For all 3-valent compositions the pH was between 5.9 and 7.1 and osmolarity was between 290-320 mOsm/kg (except one at >400 mOsm/kg). For all 6-valent compositions the pH was between 5.5 and 6.8 and osmolarity was between 260-320 mOsm/kg (except one at >500 mOsm/kg). A buffer control had pH 7.3 and 276 mOsm/kg.

(15) The integrity and immunogenicity of the combined antigens were also tested. None of antigens showed an altered analytical profile after being formulated as combinations i.e. the antigens and adjuvants are physically compatible together.

(16) With aluminium hydroxide alone all antigens adsorbed well to the adjuvant. With aluminium hydroxide and compound T (i.e. aluminium hydroxide which had been pre-mixed with compound T to permit adsorption for formation of a stable adjuvant complex; Al-T hereafter) all antigens adsorbed well, except that TT, pertactin and PT were partially desorbed.

(17) With the PLG adjuvant the diphtheria and tetanus toxoids were unadsorbed but pertussis toxoid was adsorbed.

(18) Mice (female Balb/c, 4 weeks old) were immunised intramuscularly with 100 l of each composition (i.e. human dose volume) at days 0 and 28. Sera were collected 14 days after each injection. After the second immunisation IgG antibody titers were as follows:

(19) TABLE-US-00003 Al No hydrox- Infanrix- adjuvant ide MF59 PLG Al-T 6 3-valent vaccines DT 750 21626 15693 9430 23395 TT 13120 17868 22458 15917 23131 Pertactin 639 7209 10258 3946 12857 PT 2501 8270 7212 3679 9938 FHA 3982 12057 14098 14139 23008 6-valent vaccine DT 1751 18914 13982 7658 23102 21581 TT 12729 16756 22229 13744 23267 15998 Pertactin 333 6299 9363 2912 5153 10809 PT 3069 3384 4823 3906 6484 6052 FHA 4558 7206 16201 15206 19383 11051 Hib 177 813 1266 654 2153 1269 HBsAg 1058 1598 2288 1053 4501 1113

(20) Thus for all of these antigens the inclusion of an adjuvant increased IgG antibody titers. The best titers were seen when using Al-T. The next best were with MF59, which gave better results than aluminium hydroxide alone. The titers obtained using Al-T were better for all antigens than those seen with Infanrix Hexa, except for pertactin.

(21) Furthermore, the data show that the good results achieved with the 3-valent vaccine are maintained even after IPV, Hib and HBsAg are added.

(22) IgG responses were also investigated by subclass. For most of the antigens in the 6-valent vaccines the adjuvants had little effect on IgG1 titers, but they did increase IgG2a and IgG2b titers. The best IgG2a and IgG2b titers were obtained with Al-T, and then with MF59.

(23) The increased titers seen with Al-T compared with aluminium hydroxide alone, or with the mixture of aluminium salts seen in Infanrix Hexa, mean that the total amount of aluminium per dose can be reduced while maintaining enhancement of immune responses.

(24) Reduction of Antigen Doses

(25) Experiments were designed to investigate whether the improved adjuvants could be used to reduce the amount of antigen per dose. 10-fold, 50-fold and 100-fold dilutions (relative to human dosing i.e. to deliver 1 g, 0.2 g or 0.1 g HBsAg to each mouse per 100 l dose) of the 6-valent antigen combinations were made while adjuvant concentration was maintained.

(26) Osmolarity and pH were measured (and, if necessary, adjusted) after dilution. For all 6-valent compositions the pH was between 6.1 and 7.0 and osmolarity was between 275-320 mOsm/kg. A buffer control had pH 7.3 and 285 mOsm/kg.

(27) Mice were immunised in the same way as discussed above. Total serum IgG titers after 2 immunisations were as follows:

(28) TABLE-US-00004 No adjuvant Al hydroxide MF59 Al-T 1/10 1/50 1/100 1/10 1/50 1/100 1/10 1/50 1/100 1/10 1/50 1/100 DT 459 2043 137 18357 13106 7541 17431 6003 8736 21913 16807 13724 TT 7602 7929 1700 17595 9664 5531 22791 12062 13015 23570 12237 13183 Pertactin 827 2154 341 10880 8135 4181 17159 10591 7288 17098 10748 8952 PT 3612 5645 2129 5287 3266 1068 7200 3659 5493 9051 4203 2717 FHA 2305 4161 101 8997 4471 1442 19197 5179 4492 22151 8293 3252 Hib 171 352 109 1380 796 251 3147 573 2415 3056 1440 1815 HBsAg 525 412 129 1034 685 226 4885 1103 1983 5270 1526 950

(29) Thus the presence of adjuvants allowed a dose reduction of 5-fold or 10-fold while maintaining IgG titers which are comparable or higher to unadjuvanted antigens. MF59 and AlT in particular are useful for dose sparing of antigens in this manner.

(30) Adjuvant Dosing

(31) With the 100-fold antigen dilution the amount of adjuvant was also reduced. The MF59 emulsion was mixed with antigens at a 1:1 volume ratio or at a 1:3 ratio (i.e. 1 ml of emulsion for every 3 ml of antigen, with 2 ml of buffer to maintain total volume) or at a 1:10 ratio. The Al-T complex was prepared at 3 strengths having 2 mg/ml aluminium hydroxide with either 5 g, 25 g or 100 g of compound T per dose. For comparison a 1:100 antigen dose was tested in unadjuvanted form or with aluminium hydroxide alone. A 1:100 dilution of Infanrix Hexa was also used for comparison. Osmolarity and pH were measured (and, if necessary, adjusted) after mixing (except for Infanrix Hexa). For all 6-valent compositions the pH was between 6.2 and 7.3 and osmolarity was between 270-320 mOsm/kg. A buffer control had pH 7.3 and 280 mOsm/kg.

(32) Mice were immunised as before. Total serum IgG titers after 2 immunisations were as follows:

(33) TABLE-US-00005 No Infanrx Al MF59 (v:v) Al-T (g T) adjuvant Hexa hydroxide 1:1 1:3 1:10 100 25 5 DT 584 6282 10849 7786 4094 8442 21571 20865 11788 TT 3426 5415 6857 11506 9197 11422 16041 15124 6236 Pertactin 48 3017 6053 8838 2970 2876 6158 6697 3815 PT 3351 1751 2699 4406 5072 6020 2476 2696 3079 FHA 262 7886 5626 14700 11340 10205 7369 8634 6120 Hib 126 109 310 518 517 550 936 792 390 HBsAg 88 240 369 2645 1784 1670 4062 2308 1154

(34) Thus lower amounts of MF59 and Al-T still retain good adjuvanticity and can induce higher IgG antibody titers than those induced by unadjuvanted 6-valent antigen formulations. By reducing the amount of adjuvant, while maintaining immunological efficacy, the safety profile of a vaccine can be improved which is particularly important in pediatric settings.

(35) 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.

(36) TABLE-US-00006 TABLE A antigen and Al.sup.+++ content of various marketed vaccines (per unit dose) D T Pa.sup.(1) Hib.sup.(2) IPV.sup.(3) HBsAg Vol Al.sup.+++ Pediacel 15 Lf 5 Lf 20/20/3 10 40/8/32 0.5 ml 0.33 mg Pediarix 25 Lf 10 Lf 25/25/8 40/8/32 10 g 0.5 ml 0.85 mg Pentacel 15 Lf 5 Lf 20/20/3 10 40/8/32 0.5 ml 0.33 mg Tritan.sup.x HB 30 IU 60 IU .sup.(4) 10 g 0.5 ml 0.63 mg Quinvaxem 30 IU 60 IU .sup.(4) 10 10 g 0.5 ml 0.3 mg Hexavac 30 Lf 10 Lf 25/25/ 12 40/8/32 5 g 0.5 ml 0.3 mg Boostrix 2.5 Lf 5 Lf 8/8/2.5 0.5 ml 0.39 mg Adacel 5 Lf 2 Lf 2.5/5/3 0.5 ml 0.33 mg Daptacel 15 Lf 5 Lf 10/5/3 0.5 ml 0.33 mg Pentavac 30 IU 40 IU 25/25/ 10 40/8/32 0.5 ml 0.30 mg SII QVac 20-30 Lf 5-25 Lf .sup.(4) 10 g 0.5 ml 1.25 mg TripVacHB 30 IU 60 IU .sup.(4) 10 g 0.5 ml 1.25 mg Notes: .sup.(1)Pa dose shows amounts of pertussis toxoid, then FHA, then pertactin (g). Pediacel's, Daptacel's and Adacel's Pa components also contain fimbriae types 2 and 3. .sup.(2)Hib dose shows amount of PRP capsular saccharide (g). .sup.(3)IPV dose shows amounts of type 1, then type 2, then type 3 (measured in DU). .sup.(4)Tritanrix-HepB, Quinvaxem, Trip Vac HB and SII Q-Vac include whole-cell pertussis antigens

REFERENCES

(37) [1] Vaccines. (eds. Plotkin & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0.

(38) [2] Francois et al. (2005) Pediatr Infect Dis J24:953-61.

(39) [3] Baylor et al. (2001) Vaccine 20 (Supplement 3):518-523

(40) [4] Tamm et al. (2005) Vaccine 23:1715-19.

(41) [5] WO2008/028956.

(42) [6] National Institute for Biological Standards and Control; Potters Bar, UK. www.nibsc.ac.uk

(43) [7] Sesardic et al. (2001) Biologicals 29:107-22.

(44) [8] NIBSC code: 98/560.

(45) [9] Module 1 of WHO's The immunological basis for immunization series (Galazka).

(46) [10] NIBSC code: 69/017.

(47) [11] NIBSC code: DIFT.

(48) [12] Sesardic et al. (2002) Biologicals 30:49-68.

(49) [13] NIBSC code: 98/552.

(50) [14] NIBSC code: TEFT.

(51) [15] Rappuoli et al. (1991) TIBTECH 9:232-238.

(52) [16] Nencioni et al. (1991) Infect Immun. 59(2): 625-30.

(53) [17] Ramsay et al. (2001) Lancet 357(9251):195-196.

(54) [18] Lindberg (1999) Vaccine 17 Suppl 2:S28-36.

(55) [19] Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-168.

(56) [20] Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii.

(57) [21] Goldblatt (1998) J. Med. Microbiol. 47:563-567.

(58) [22] European patent 0477508.

(59) [23] U.S. Pat. No. 5,306,492.

(60) [24] WO98/42721.

(61) [25] Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114.

(62) [26] Hermanson (1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X.

(63) [27] WO96/40242.

(64) [28] Vanlandschoot et al. (2005) J Gen Virol 86:323-31.

(65) [29] WO2007/054820.

(66) [30] WO03/066094.

(67) [31] Liao et al. (2012) J Infect Dis. 205:237-43.

(68) [32] Verdijk et al. (2011) Expert Rev Vaccines. 10:635-44.

(69) [33] Module 6 of WHO's The immunological basis for immunization series (Robertson)

(70) [34] W.H.O. Tech. Rep. Ser. 594:51, 1976.

(71) [35] WO03/080678.

(72) [36] Glode et al. (1979) J Infect Dis 139:52-56

(73) [37] WO94/05325; U.S. Pat. No. 5,425,946.

(74) [38] Arakere & Frasch (1991) Infect. Immun. 59:4349-4356.

(75) [39] Michon et al. (2000) Dev. Biol. 103:151-160.

(76) [40] Rubinstein & Stein (1998) J. Immunol. 141:4357-4362.

(77) [41] WO2005/033148

(78) [42] WO2005/000347.

(79) [43] WO02/058737.

(80) [44] WO03/007985.

(81) [45] WO2007/000314.

(82) [46] WO2007/000322.

(83) [47] Giuliani et al. (2006) Proc Natl Acad Sci USA 103(29):10834-9.

(84) [48] WO99/57280.

(85) [49] Masignani et al. (2003) J Exp Med 197:789-799.

(86) [50] Welsch et al. (2004) J Immunol 172:5605-15.

(87) [51] Hou et al. (2005) J Infect Dis 192(4):580-90.

(88) [52] WO03/063766.

(89) [53] Fletcher et al. (2004) Infect Immun 72:2088-2100.

(90) [54] Zhu et al. (2005) Infect Immun 73(10):6838-45.

(91) [55] WO2004/048404

(92) [56] Serruto et al. (2010) PNAS USA 107:3770-5.

(93) [57] Tettelin et al. (2000) Science 287:1809-15.

(94) [58] WHO Technical Report Series No. 927, 2005. Pages 64-98.

(95) [59] US-2008/0102498.

(96) [60] US-2006/0228381.

(97) [61] US-2007/0231340.

(98) [62] US-2007/0184072.

(99) [63] US-2006/0228380.

(100) [64] WO2008/143709.

(101) [65] Research Disclosure, 453077 (January 2002)

(102) [66] EP-A-0378881.

(103) [67] EP-A-0427347.

(104) [68] WO93/17712

(105) [69] WO94/03208.

(106) [70] WO98/58668.

(107) [71] EP-A-0471177.

(108) [72] WO91/01146

(109) [73] Falugi et al. (2001) Eur J Immunol 31:3816-3824.

(110) [74] Baraldo et al. (2004) Infect Immun 72(8):4884-7.

(111) [75] EP-A-0594610.

(112) [76] Ruan et al. (1990) J Immunol 145:3379-3384.

(113) [77] WO00/56360.

(114) [78] Kuo et al. (1995) Infect Immun 63:2706-13.

(115) [79] Michon et al. (1998) Vaccine. 16:1732-41.

(116) [80] WO02/091998.

(117) [81] WO01/72337

(118) [82] WO00/61761.

(119) [83] WO00/33882

(120) [84] WO2007/071707

(121) [85] Bethell G. S. et al., J. Biol. Chem., 1979, 254, 2572-4

(122) [86] Hearn M. T. W., J. Chromatogr., 1981, 218, 509-18

(123) [87] WO2007/000343.

(124) [88] Mol. Immunol., 1985, 22, 907-919

(125) [89] EP-A-0208375

(126) [90] WO00/10599

(127) [91] Gever et al., Med. Microbiol. Immunol, 165: 171-288 (1979).

(128) [92] U.S. Pat. No. 4,057,685.

(129) [93] U.S. Pat. Nos. 4,673,574; 4,761,283; 4,808,700.

(130) [94] U.S. Pat. No. 4,459,286.

(131) [95] U.S. Pat. No. 5,204,098

(132) [96] U.S. Pat. No. 4,965,338

(133) [97] U.S. Pat. No. 4,663,160.

(134) [98] US-2007/0184071.

(135) [99] Jodar et al. (2003) Vaccine 21:3265-72.

(136) Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).

(137) Clausi et al. (2008) J Pharm Sci DOI 10.1002/jps.21390.

(138) International patent application PCT/US2011/050231.

(139) U.S. patent application 61/316,551.

(140) U.S. patent application 61/379,126.

(141) WO2011/027222.

(142) WO2011/024072.

(143) WO2010/144734.

(144) WO90/14837.

(145) Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203.

(146) Podda (2001) Vaccine 19: 2673-2680.

(147) Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan.

(148) WO2008/043774.

(149) Allison & Byars (1992) Res Immunol 143:519-25.

(150) Hariharan et al. (1995) Cancer Res 55:3486-9.

(151) US-2007/014805.

(152) WO2007/080308.

(153) WO95/11700.

(154) U.S. Pat. No. 6,080,725.

(155) WO2005/097181.

(156) WO2006/113373.

(157) U.S. Pat. No. 6,630,161.

(158) Nony et al. (2001) Vaccine 27:3645-51.

(159) WO01/41800.

(160) Dupuis et al. (1999) Vaccine 18:434-9.

(161) Immunopotentiators in Modern Vaccines (2006) Schijns and O'Hagan (eds.) ISBN: 0-12-088403-8.