Synthetic and natural TLR4 agonists as safe and effective vaccine adjuvants

Sub-Cellular Biochemistry

Volumn 53, Issue , 2010, Pages 303-321

  Fox, Christopher B ^a   Friede, Martin ^a   Reed, Steven G ^a   Ireton, Gregory C ^b  

^a Infectious Disease Research Institute ^*

^b INFECTIOUS DISEASE RESEARCH INSTITUTE   (United States)

Author keywords

Adjuvant formulation; Glucopyranosyl lipid adjuvant; Lipopolysaccharide; Monophosphoryl lipid; TLR4 agonist

Indexed keywords

MAMMALIA;

IMMUNOLOGICAL ADJUVANT; LIPOPOLYSACCHARIDE; TOLL LIKE RECEPTOR 4;

ANIMAL; ARTICLE; CARBOHYDRATE ANALYSIS; CHEMICAL STRUCTURE; CHEMISTRY; CONFORMATION; DRUG POTENTIATION; HUMAN; IMMUNOLOGY; MOLECULAR GENETICS;

ADJUVANTS, IMMUNOLOGIC; ANIMALS; CARBOHYDRATE CONFORMATION; CARBOHYDRATE SEQUENCE; HUMANS; LIPOPOLYSACCHARIDES; MOLECULAR SEQUENCE DATA; MOLECULAR STRUCTURE; TOLL-LIKE RECEPTOR 4;

EID: 77956630701     PISSN: 03060225     EISSN: None     Source Type: Journal    
DOI: 10.1007/978-90-481-9078-2_14     Document Type: Article

References (89)

1. Adams, J.U. Hypertension's shot in the arm. Nature Biotech 26 (2008) 1327-1329
2. Akira, S., Takeda, K. Toll-like receptor signaling. Nat Rev Immunol 4 (2004) 499-511
3. Akira, S., Uematsu, S., Takeuchi, O. Pathogen recognition and innate immunity. Cell 124 (2006) 783-801.
4. Alexander, C., Rietschel, E.T. Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res 7 (2001) 167-202.
5. Alving, C.R., Matyas, G.R. Design and selection of vaccine adjuvants: principles and practice. In Kaufmann, S.H.E., Lambert, P.H. (eds), The Grand Challenge for the Future: Vaccines for Poverty-Related Diseases from Bench to Field. Birkhauser Verlag, Basel, Switzerland (2005), pp. 99-118
6. Alving, C.R., Rao, M. Lipid A and liposomes containing lipid A as antigens and adjuvants. Vaccine 26 (2008) 3036-3045.
7. Anderson, R.C., Fox, C.B., Dutill, T.S., Shaverdian, N., Evers, T.L., Poshusta, G.R., Chesko, J., Coler, R.N., Friede, M., Reed, S.G., Vedvick, T.S. Physicochemical characterization and biological activity of synthetic TLR4 agonist formulations. Coll Surf B: Biointerfaces 75 (2010) 123-132.
8. Atanackovic, D., Altorki, N.K., Cao, Y., Ritter, E., Ferrara, C.A., Ritter, G., Hoffman, E.W., Bokemeyer, C., Old, L.J., Gnjatic, S. Booster vaccination of cancer patients with MAGE-A3 protein reveals long-term immunological memory or tolerance depending on priming. Proc Nat Acad Sci USA 105 (2008) 1650-1655.
9. Baldridge, J.R., Crane, R.T. Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines. Methods 19 (1999) 103-107.
10. Baldwin, S.L., Bertholet, S., Kahn, M., Zharkikh, I., Ireton, G.C., Vedvick, T.S., Reed, S.G., Coler, R.N. Intradermal immunization improves protective efficacy of a novel TB vaccine candidate. Vaccine 27 (2009a) 3063-3071.
11. Baldwin, S.L., Shaverdian, N., Goto, Y., Duthie, M.S., Raman, V.S., Evers, T., Mompoint, F., Vedvick, T.S., Bertholet, S., Coler, R.N., Reed, S.G. Enhanced humoral and Type 1 cellular immune responses with Fluzone adjuvanted with a synthetic TLR4 agonist formulated in an emulsion. Vaccine 27 (2009b) 5956-5963.
12. Barton, G.M., Kagan, J.C. A cell biological view of Toll-like receptor function: regulation through compartmentalization. Nat Rev Immunol 9 (2009) 535-542
13. Bertholet, S., Goto, Y., Carter, L., Bhatia, A., Howard, R.F., Carter, D., Coler, R.N., Vedvick, T.S., Reed, S.G. Optimized subunit vaccine protects against experimental leishmaniasis. Vaccine 27 (2009) 7036-7045.
14. Beutler, B., Rietschel, E.T. Innate immune sensing and its roots: the story of endotoxin. Nat Rev Immunol 3 (2003) 169-176
15. Beutler, B.A. TLRs and innate immunity. Blood 113 (2009) 1399-1407.
16. Bodewes, R., Geelhoed-Mieras, M.M., Heldens, J.G.M., Glover, J., Lambrecht, B.N., Fouchier, R.A.M., Osterhaus, A.D.M.E., Rimmelzwaan, G.F. The novel adjuvant CoVaccineHTTM increases the immunogenicity of cell-culture derived influenza A/H5N1 vaccine and induces the maturation of murine and human dendritic cells in vitro. Vaccine 27 (2009) 6833-6839.
17. Brandenburg, K., Lindner, B., Schromm, A., Koch, M.H., Bauer, J., Merkli, A., Zbaeren, C., Davies, J.G., Seydel, U. Physicochemical characteristics of triacyl lipid A partial structure OM-174 in relation to biological activity. Eur J Biochem 267 (2000) 3370-3377
18. Caglar, C., Casella, R.C., Eaves, C.A., Matsuzawa, A., Ichijo, H., Mitchell, T.C. Selective activation of the p38 MAPK pathway by synthetic monophosphoryl lipid A. J Biol Chem 284 (2009) 31982-31991.
19. Childers, N.K., Miller, K.L., Tong, G., Llarena, J.C., Greenway, T., Ulrich, J.T., Michalek, S.M. Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antigen Infect Immun 68 (2000) 5509-5516
20. Choe, J., Kelker, M.S., Wilson, I.A. Crystal structure of human toll-like receptor 3 (TLR3) ectodomain. Science 309 (2005) 581-585
21. Cohen, J., Druilhe, P. Immunogenic compositions comprising liver stage malarial antigens. WIPO WO/2002/038176 (2002)
22. Crane, R.T. Aqueous immunologic adjuvant compositions of monophosphoryl lipid A. U.S. Patent 6,491,919 B2 (2002)
23. Davidsen, J., Andersen, P., Rosenkrands, I. Compositions and methods for stabilizing lipid based adjuvant formulations using glycolipids. U.S. Patent US 2006/0008519 A1 (2006)
24. Elliott, G.T., McLeod, R.A., Perez, J., von Eschen, K.B. Interim results of a phase II multicenter clinical trial evaluating the activity of a therapeutic allogeneic melanoma vaccine (theraccine) in the treatment of disseminated malignant melanoma. Semin Surg Oncol 9 (1993) 264-272
25. Evans, J.T., Cluff, C.W., Johnson, D.A., Lacy, M.J., Persing, D.H., Baldridge, J.R. Enhancement of antigen-specific immunity via the TLR4 ligands MPLTM adjuvant and Ribi.529. Expert Rev Vaccines 2 (2003) 219-229
26. Floyd, A.G. Top ten considerations in the development of parenteral emulsions. Pharm Sci Tech Today 2 (1999) 134-143
27. Fries, L.F., Gordon, D.M., Richards, R.L., Egan, J.E., Hollingdale, M.R., Gross, M., Silverman, C., Alving, C.R. Liposomal malaria vaccine in humans: a safe and potent adjuvant strategy. Proc Natl Acad Sci USA 89 (1992a) 358-362
28. Fries, L.F., Gordon, D.M., Richards, R.L., Egan, J.E., Hollingdale, M.R., Gross, M., Silverman, C., Alving, C.R. Liposomal malaria vaccine in humans: a safe and potent adjuvant strategy. Proc Nat Acad Sci USA 89 (1992b) 358-362
29. Galanos, C., Luderitz, O., Freudenberg, M., Brade, L., Schade, U., Rietschel, E.T., Kusumoto, S., Shiba, T. Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A. Eur J Biochem 160 (1986) 55-59
30. Garcon, N. Development and evaluation of AS04, a novel and improved adjuvant system contain- ingMPL andaluminum salt.InSchijns, V.,O'Hagan, D. (eds), Immunopotentiators in Modern Vaccines. Academic Press, London (2005), pp. 161-178
31. Garidel, P., Rappolt, M., Schromm, A.B., Howe, J., Lhner, K., Andra, J., Koch, M.H., Brandenburg, K. Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity. Biochim Biophys Acta 1715(2005) 122-131.
32. Gawchik, S.M., Saccar, C.L. Pollinex Quattro Tree: Allergy vaccine. Expert Opin Biol Ther 9 (2009) 377-382.
33. Hajjar, A.M., Ernst, R.K., Tsai, J.H., Wilson, C.B., Miller, S.I. Human Toll-like receptor 4 recognizes host-specific LPS modifications. Nat Immunol 3 (2002) 354-359.
34. Hui, G.S., Hashimoto, C.N. Adjuvant formulations possess differing efficacy in the potentiation of antibody and cell mediated responses to a human malaria vaccine under selective immune genes knockout environment. Int Immunopharm 8 (2008) 1012-1022.
35. Ishizaka, S.T., Hawkins, L.D. E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant. Exp Rev Vaccines 6 (2007) 773-784.
36. Janeway, C.A., Jr., Medzhitov, R. Innate immune recognition. Annu Rev Immunol 20 (2002) 197-216
37. Jiang, Z.H., Bach, M.V., Budzynski, W.A., Krantz, M.J., Koganty, R.R., Longenecker, B.M. Lipid A structures containing novel lipid moieties: Synthesis and adjuvant properties. Bioorg Med Chem Lett 12 (2002) 2193-2196.
38. Jiang, Z.H., Budzynski, W.A., Qiu, D., Yalamati, D., Koganty, R.R. Monophosphoryl lipid A analogues with varying 3-O-substitution: Synthesis and potent adjuvant activity. Carbohydr Res 342 (2007) 784-796
39. Johnson, A. Molecular adjuvants and immunomodulators: New approaches to immunization. Clin Microbiol Rev 7 (1994) 277-289
40. Johnson, A.G. Adjuvant action of bacterial endotoxins on the primary antibody response. In Landy, M., Braun, W. (eds), Bacterial Endotoxins. Rutgers University Press, New Brunswick, CT (1964), pp. 252-262.
41. Johnson, A.G., Gains, S., Landy, M. Studies on the O antigen of Salmonella ryphosa V. Enhancement of antibody response to protein antigens by purified lipopolysaccharide. J Exp Med 103 (1956) 225-246
42. Johnson, D.A., Keegan, D.S., Sowell, C.G., Livesay, M.T., Johnson, C.L., Taubner, L.M., Harris, A., Myers, K.R., Thompson, J.D., Gustafson, G.L., Rhodes, M.J., Ulrich, J.T., Ward, J.R., Yorgensen, Y.M., Cantrell, J.L., Brookshire, V.G. 3-O-Desacyl monophosphoryl lipid A derivatives: Synthesis and immunostimulant activities. J Med Chem 42 (1999) 4640-4649
43. Jurgens, G., Muller, M., Koch, M.H., Brandenburg, K. Interaction of hemoglobin with enterobac-terial lipopolysaccharide and lipid A. Physicochemical characterization and biological activity. Eur J Biochem 268 (2001) 4233-4242.
44. Kim, H.M., Park, B.S., Kim, J.I., Kim, S.E., Lee, J., Oh, S.C., Enkhbayar, P., Matsushima, N., Lee, H., Yoo, O.J., Lee, J.O. Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130 (2007) 906-917.
45. Kobayashi, M., Saitoh, S., Tanimura, N., Takahashi, K., Kawasaki, K., Nishijima, M., Fujimoto, Y., Fukase, K., Akashi-Takamura, S., Miyake, K. Regulatory roles for MD-2 and TLR4 in ligand-induced receptor clustering. J Immunol 176 (2006) 6211-6518.
46. Kotani, S., Takada, H., Takahashi, I., Ogawa, T., Tsujimoto, M., Shimauchi, H., Ikeda, T., Okamura, H., Tamura, T., Harada, K. Immunobiological activities of synthetic lipid A analogs with low endotoxicity. Infect Immun 54 (1986) 673-682.
47. Kumar, V.V. Complementary molecular shapes and additivity of the packing parameter of lipids. Proc Natl Acad Sci USA 88 (1991) 444-448.
48. Kumazawa, Y., Matsuura, M., Homma, J.Y., Nakatsuru, Y., Kiso, M., Hasegawa, A. B cell activation and adjuvant activities of chemically synthesized analogues of the nonreducing sugar moiety of lipid A. Eur J Immunol 15 (1985) 199-201.
49. Kusumoto, S., Fukase, K., Fukase, Y., Kataoka, M., Yoshizaki, H., Sato, K., Oikawa, M., Suda, Y. Structural basis for endotoxic and antagonistic activities: investigation with novel synthetic lipid A analogs. J Endotoxin Res 9 (2003) 361-366.
50. Lell, B., Agnandji, S., von Glasenapp, I., Haertle, S., Oyakhiromen, S., Issifou, S., Vekemans, J., Leach, A., Lievens, M., Dubois, M.C., Demoitie, M.A., Carter, T., Villafana, T., Ballou, W.R., Cohen, J., Kremsner, P.G. A randomized trial assessing the safety and immunogenicity of AS01 and AS02 adjuvanted RTS,S malaria vaccine candidates in children in Gabon. PLoS ONE 4 (2009) e7611.
51. Liu, F., Liu, D. Long-circulating emulsions (oil-in-water) as carriers for lipophilic drugs. Pharm Res 12 (1995) 1060-1064
52. Liu, L., Botos, I., Wang, Y., Leonard, J.N., Shiloach, J., Segal, D.M., Davies, D.R. Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science 320 (2008) 379-381
53. Martin, O.R., Zhou, W., Wu, X., Front-Deschamps, S., Moutel, S., Schindl, K., Jeandet, P., Zbaeren, C., Bauer, J.A. Synthesis and immunobiological activity of an original series of acyclic lipid A mimics based on a pseudodipeptide backbone. J Med Chem 49 (2006) 6000-6014.
54. Mata-Haro, V., Cekic, C., Martin, M., Chilton, P.M., Casella, C.R., Mitchell, T.C. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science 316 (2007) 1628-1632.
55. Matsushima, N., Tanaka, T., Enkhbayar, P., Mikami, T., Taga, M., Yamada, K., Kuroki, Y. Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors. BMC Genomics 8 (2007) 124
56. Matyas, G.R., Wassef, N.M., Rao, M., Alving, C.R. Induction and detection of antibodies to squalene. J Immunol Methods 245 (2000) 1-14.
57. McClements, D.J. Critical review of techniques and methodologies for characterization of emulsion stability. Crit Rev Food Sci Nutri 47 (2007) 611-649.
58. McClements, D.J., Decker, E.A. Lipid oxidation in oil-in-water emulsions: impact of molecular environment on chemical reactions in heterogeneous food systems. J Food Sci 65 (2000) 1270-1282.
59. Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature 449 (2007) 819-826.
60. Medzhitov, R., Preston-Hurlburt, P., Janeway, C.A., Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388 (1997) 394-397.
61. Mitchell, M.S., Harel, W., Kempf, R.A., Hu, E., Kan-Mitchell, J., Boswell, W.D., Dean, G., Stevenson, L. Active-specific immunotherapy for melanoma. J Clin Oncol 8 (1990) 856-869
62. Miyake, K. Roles for accessory molecules in microbial recognition by Toll-like receptors. J Endotoxin Res 12 (2006) 195-204
63. Morefield, G.L., Hawkins, L.D., Ishizaka, S.T., Kissner, T.L., Ulrich, R.G. Synthetic Toll-like receptor 4 agonist enhances vaccine efficacy in an experimental model of toxic shock syndrome.Clin Vaccine Immunol 14 (2007) 1499-1504
64. Mueller, M., Lindner, B., Kusumoto, S., Fukase, K., Schromm, A.B., Seydel, U. Aggregates are the biologically active units of endotoxin. J Biol Chem 279 (2004) 26307-26313
65. Myers, K., Truchot, A., Ward, J., Hudson, Y., Ulrich, J. A critical determinant of lipid A endo- toxic activity. In Nowotny, A., Spitzler, J., Ziegler, E. (eds), Cellular and Molecular Aspects ofEndotoxin Reactions. Elsevier, Amsterdam (1990), pp. 145-156
66. Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y., Akira, S., Kitamura, T., Kosugi, A., Kimoto, M., Miyake, K. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 3 (2002) 667-672.
67. Norden, T.P., Siekmann, B., Lundquist, S., Malmsten, M. Physicochemical characterisation of a drug-containing phospholipid-stabilised o/w emulsion for intravenous administration. Eur J Pharm Sci 13 (2001) 393-401.
68. Ohto, U., Fukase, K., Miyake, K., Satow, Y. Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa. Science 316 (2007) 1632-1634.
69. Owusu-Agyei, S., Ansong, D., Asante, K., Owusu, S.K., Owusu, R., Brobby, N.A.W., Dosoo, D., Akoto, A.O., Osei-Kwakye, K., Adjei, E.A., Boahen, K.O., Sylverken, J., Adjei, G., Sambian, D., Apanga, S., Kayan, K., Vekemans, J., Ofori-Anyinam, O., Leach, A., Lievens, M., Demoitie, M.A., Dubois, M.C., Cohen, J., Ballou, W.R., Savarese, B., Chandramohan, D., Gyapong, J.O., Milligan, P., Antwi, S., Agbenyega, T., Greenwood, B., Evans, J. Randomized controlled trial of RTS,S/AS02D and RTS,S/AS01E malaria candidate vaccines given according to different schedules in Ghanaian children. PLoS One 4 (2009) e7302.
70. Paavonen, J., Naud, P., Salmeron, J., Wheeler, C.M., Chow, S.N., Apter, D., Kitchener, H., Castellsague, X., Teixeira, J.C., Skinner, S.R., Hedrick, J., Jaisamram, U., Limson, G., Garland, S., Szarewski, A., Romanowski, B., Aoki, F.Y., Schwarz, T.F., Poppe, W.A., Bosch, F.X., Jenkins, D., Hardt, K., Zahaf, T., Descamps, D., Struyf, F., Lehtinen, M., Dubin, G., Group, H.P.S., Greenacre, M. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet 374 (2009) 301-314.
71. Park, B.S., Song, D.H., Kim, H.M., Choi, B.S., Lee, H., Lee, J.O. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 458 (2009) 1191-1195.
72. Pasare, C., Medzhitov, R. Toll-like receptors: Linking innate and adaptive immunity. Microbes Infect 6 (2004) 1382-1387
73. Pichyangkul, S., Gettayacamin, M., Miller, R.S., Lyon, J.A., Angov, E., Tongtawe, P., Ruble, D.L., Heppner, D.G., Jr., Kester, K.E., Ballou, W.R., Diggs, C.L., Voss, G., Cohen, J.D., Walsh, D.S. Pre-clinical evaluation of the malaria vaccine candidate P. falciparum MSP142 formulated with novel adjuvants or with alum. Vaccine 22 (2004) 3831-3840.
74. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., Beutler, B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282 (1998) 2085-2088.
75. Qureshi, N., Takayama, K., Ribi, E. Purification and structural determination of non-toxic lipid-A from lipopolysaccharide of Salmonella typhimurium. J Biol Chem 257 (1982) 11808-11815.
76. Raetz, C.R., Whitfield, C. Lipopolysaccharide endotoxins. Annu Rev Biochem 71 (2002) 635-700.
77. Reed, S.G., Bertholet, S., Coler, R.N., Friede, M. New horizons in adjuvants for vaccine development. Trends Immunol 30 (2009) 23-32.
78. Ribi, E., Parker, R., Strain, S., Mizuno, Y., Nowomy, A., Von Eschen, K., Cantrell, J., McLaughlin, C., Hwang, K., Goren, M. Peptides as requirement for immunotherapy of the guinea-pig line 10 tumor with endotoxins. Cancer Immunol Immunother 12 (1979) 91-96.
79. Richards, R. L., Rao, M., Wassef, N.M., Glenn, G.M., Rothwell, S.W., Alving, C.R. Liposomescontaining Lipid A serve as an adjuvant for induction of antibody and cytotoxic T-cell responses against RTS,S malaria antigen. Infect Immun 66 (1998) 2859-2865
80. Rietschel, E.T., Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppnow, H., Ulmer, A.J., Zahringer, U., Seydel, U., Di Padova, F., et al. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J 8 (1994) 217-225
81. Seydel, U., Oikawa, M., Fukase, K., Kusumoto, S., Brandenburg, K., Intrinsic conformation of lipid A is responsible for agonistic and antagonistic activity. Eur J Biochem 267 (2000) 3032-3039.
82. Seydel, U., Schromm, A.B., Brade, L., Gronow, S., Andra, J., Muller, M., Koch, M.H., Fukase, K., Kataoka, M., Hashimoto, M., Kusumoto, S., Brandenburg, K. Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity. FEBS J 272 (2005) 327-340.
83. Simberg, D., Weisman, S., Talmon, Y., Barenholz, Y. DOTAP (and other cationiclipids): chemistry, biophysics, and transfection. Crit Rev Ther Drug Carr Syst 21 (2004) 257-317.
84. Stover, A.G., Da Silva Correia, J., Evans, J.T., Cluff, C.W., Elliott, M.W., Jeffery, E.W., Johnson, D. A., Lacy, M.J., Baldridge, J.R., Probst, P., Ulevitch, R.J., Persing, D.H., Hershberg, R.M. Structure-activity relationship of synthetic toll-like receptor 4 agonists. J Biol Chem 279 (2004) 4440-4449.
85. Tsujimoto, M., Kotani, S., Okunaga, T., Kubo, T., Takada, H., Kubo, T., Shiba, T., Kusumoto, S., Takahashi, T., Goto, Y. Enhancement of humoral immune responses against viral vaccines by a non-pyrogenic 6-O-acylmuramyldipeptide and synthetic low toxicity analogues of lipid A. Vaccine 7 (1989) 39-48.
86. Ulmer, A.J., Heine, H., Feist, W., Kusumoto, S., Kusama, T., Brade, H., Schade, U., Rietschel, E.T., Flad, H.D. Biological activity of synthetic phosphonooxyethyl analogs of lipid A and lipid A partial structures. Infect Immun 60 (1992) 3309-3314.
87. Ulrich, J., Myers, K. Monophosphoryl lipid A as an adjuvant. In Powell, M., Newman, M. (eds), Vaccine Design: The subunit and adjuvant approach. Plenum Press, New York (1995), pp. 495-524.
88. Zhao, B., Rong, Y.-Z., Huang, X.-H., Shen, J.-S. Experimental and theoretical study on the structure and electronic spectra of imiquimod and its synthetic intermediates. Bioorg Med Chem Lett 17 (2007) 4942-4946.
89. Zhu, D., Huang, S., Gebregeorgis, E., McClellan, H., Dai, W., Miller, L., Saul, A. Development of a direct alhydrogel formulation immunoassay (DAFIA). J Immunol Methods 344 (2009) 73-78.