咪喹莫特作为佐剂的透皮免疫效果研究
摘要
皮肤是人体的最大的组织和器官，它的表面积接近2m~2。皮肤也是一个具有独特免疫功能并与全身免疫系统密切相关的组织器官。
德国的医学学生Langerhans P于1868年首次发现并描述了皮肤中的抗原递呈细胞，后被命名为朗格汉斯细胞(Langerhans cell，LC)。未成熟的朗格汉斯细胞具有较强吞噬处理抗原能力，并沿淋巴管迁移至近侧引流淋巴结(draining lymph nodes，DLN)的T细胞区并分化成熟而具有很强的抗原递呈(包括交叉递呈)能力。
透皮免疫(Transcutaneous immunization，TCI)是一种新的疫苗接种方法。它通过在皮肤表面局部应用免疫佐剂和疫苗抗原来使机体产生全身性免疫反应。通过透皮剂等技术手段，使疫苗抗原进入表皮中并被表皮朗格汉斯细胞吞噬，在佐剂的作用下，迅速引发全身性的免疫反应，从而达到免疫效果。
2004年Karande等发现了透皮剂聚氧乙烯十二烷基醚硫酸钠(sodium laureth sulfate，SLA)+苯基哌嗪(phenyl piperazine，PP)的组合，该组合能够有效促进分子量小于1～10kD的生物大分子的透皮，并不诱导皮肤损伤。SLA+PP的组合能够在离体的皮肤中最大限度的促进肝素、促黄体(生成)激素-释放激素等生物大分子的透皮；在小鼠皮肤上的实验也证实醋酸亮丙瑞林能够在促透剂的作用下进入小鼠体内。
在研究人类单纯疱疹病毒的治疗时发现imiquimod具有很强的抗病毒和抗肿瘤活性，美国3M公司在1997年将其上市，制成5％的外用软膏剂(商品名Aldara)。咪喹莫特可以刺激单核细胞、巨噬细胞、树突状细胞产生以干扰素IFN-α、肿瘤坏死因子TNF-α、白介素IL-1、6、8、10、12为代表的多种细胞因子。我们用咪喹莫特免疫刺激特性作为透皮佐剂，研究亚单位疫苗、病毒疫苗、DNA疫苗三种疫苗的透皮免疫效果。
聚氧乙烯十二烷基醚硫酸钠和苯基哌嗪混合之后溶于等体积PBS缓冲液和无水乙醇混合液中，制成透皮剂。将其分别与HBV亚单位抗原、HBsAg DNA疫苗、灭活的HAV抗原混合后，涂抹于小鼠脱毛的背部皮肤表面，同时涂抹咪喹莫特作为佐剂，分别在2、4、6、8、12、20周用ELISA法检测血清中的抗体水平。在透皮免疫2周后即可在用咪喹莫特作为佐剂的HBV亚单位抗原组和DNA疫苗组中用ELISA法检测到血清中的抗体，平均分别为1：19.1和1：30.3；抗体滴度在8周达到最高水平，平均分别为1：105.6和1：105.6；无佐剂组在4周才检测到抗体，抗体滴度平均为1：3.31和1：1.82。结论：咪喹莫特能高效迅速诱导HBV亚单位抗原和DNA疫苗透皮免疫的抗体产生。
Human's skin has a large surface area. It is the largest tissue and organ of person.Skin has been regarded as a organ that has peculiar immune function and keeps closecorrelation with immunity system.
Langerhans cells are paradigmatic dendriticcells, described in 1868 by a youngmedical student, Paul Langerhans in Berlin. Langerhans cells are present withepithelial cells in the epidermis, bronchi and mucosae. After antigenic challenge,Langerhans cells migrate into the T cell areas of proximal lymph nodes where they actas professional antigen-presenting cells.
Transcutaneous immunization (TCI) is a novel strategy for administering adjuvantand antigen to the skin surface. The adjuvant and antigen applied using TCIapparently target Langerhans cells in the skin, eliciting systemic antibodies, includingrobust mucosal IgG and secretory IgA responses in both mice and humans andprotective immunity against mucosal challenge with toxin or live virus.
Karande describe particular mixtures of penetration enhancers that increase skinpermeability to macromolecules (＜1-10 kDa) by up to＜100-fold without inducingskin irritation. In vitro experiments demonstrated that the mixtures deliveredmacromolecular drugs, including heparin, leutinizing hormone releasing hormone(LHRH) and oligonulceotides across the skin. In vivo experiments on hairless ratswith leuprolide acetate confirmed the potency and safety of one such mixture, sodiumlaureth sulfate (SLA) and phenylpiperazine (PP).
Imiquimod, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (also knownas R-837 and S-26308), is a nonnucleoside heterocyclic amine that belongs to a classof products known as immune-response modifiers. In vivo studies have demonstratedthat imiquimod is a potent inducer of alpha IFN(IFN-a), tumor necrosis factor alpha(TNF-a), and interleukin-1,6,8,10,12 (IL-1,6,8,10,12) and that it has adjuvantproperties greater than those of Freund's complete adjuvant. In animal models,imiquimod has been demonstrated to have potent antiviral and antitumor effects.
We use the imiquimod as adjuvant to investigate the adjuvantal effect of imiquimod on transcutaneous immunization for subunit antigen, virus antigen and DNA vaccine.
SLA: PP (weight fractions,0.7:0.3;total concentration of 0.5%(wt/vol)) dissolves in1:1 phosphate buffered saline(PBS):ethanol. This solution admixes with HBV subunitantigen, HAV antigen and HBsAg DNA vaccine distinctively. The admixtures werebesmeared on the depilated mouse back and then the adjuvant imiquimod wasbesmeared on the back. At 2, 4, 6, 8, 12, 20 weeks post inoculation. The specificHBsAg antibody were detected early at two weeks after transcutaneous immunization.The titers reached the highest level at 8 weeks. The array without imiquimod coulddetect antibody only at 4 weeks post immunization.
Conclusion The adjuvant imiquimod can enhance specific antibody levelsignificantly and quickly for HBsAg and HBsAg DNA vaccine.
德国的医学学生Langerhans P于1868年首次发现并描述了皮肤中的抗原递呈细胞，后被命名为朗格汉斯细胞(Langerhans cell，LC)。未成熟的朗格汉斯细胞具有较强吞噬处理抗原能力，并沿淋巴管迁移至近侧引流淋巴结(draining lymph nodes，DLN)的T细胞区并分化成熟而具有很强的抗原递呈(包括交叉递呈)能力。
透皮免疫(Transcutaneous immunization，TCI)是一种新的疫苗接种方法。它通过在皮肤表面局部应用免疫佐剂和疫苗抗原来使机体产生全身性免疫反应。通过透皮剂等技术手段，使疫苗抗原进入表皮中并被表皮朗格汉斯细胞吞噬，在佐剂的作用下，迅速引发全身性的免疫反应，从而达到免疫效果。
2004年Karande等发现了透皮剂聚氧乙烯十二烷基醚硫酸钠(sodium laureth sulfate，SLA)+苯基哌嗪(phenyl piperazine，PP)的组合，该组合能够有效促进分子量小于1～10kD的生物大分子的透皮，并不诱导皮肤损伤。SLA+PP的组合能够在离体的皮肤中最大限度的促进肝素、促黄体(生成)激素-释放激素等生物大分子的透皮；在小鼠皮肤上的实验也证实醋酸亮丙瑞林能够在促透剂的作用下进入小鼠体内。
在研究人类单纯疱疹病毒的治疗时发现imiquimod具有很强的抗病毒和抗肿瘤活性，美国3M公司在1997年将其上市，制成5％的外用软膏剂(商品名Aldara)。咪喹莫特可以刺激单核细胞、巨噬细胞、树突状细胞产生以干扰素IFN-α、肿瘤坏死因子TNF-α、白介素IL-1、6、8、10、12为代表的多种细胞因子。我们用咪喹莫特免疫刺激特性作为透皮佐剂，研究亚单位疫苗、病毒疫苗、DNA疫苗三种疫苗的透皮免疫效果。
聚氧乙烯十二烷基醚硫酸钠和苯基哌嗪混合之后溶于等体积PBS缓冲液和无水乙醇混合液中，制成透皮剂。将其分别与HBV亚单位抗原、HBsAg DNA疫苗、灭活的HAV抗原混合后，涂抹于小鼠脱毛的背部皮肤表面，同时涂抹咪喹莫特作为佐剂，分别在2、4、6、8、12、20周用ELISA法检测血清中的抗体水平。在透皮免疫2周后即可在用咪喹莫特作为佐剂的HBV亚单位抗原组和DNA疫苗组中用ELISA法检测到血清中的抗体，平均分别为1：19.1和1：30.3；抗体滴度在8周达到最高水平，平均分别为1：105.6和1：105.6；无佐剂组在4周才检测到抗体，抗体滴度平均为1：3.31和1：1.82。结论：咪喹莫特能高效迅速诱导HBV亚单位抗原和DNA疫苗透皮免疫的抗体产生。
Human's skin has a large surface area. It is the largest tissue and organ of person.Skin has been regarded as a organ that has peculiar immune function and keeps closecorrelation with immunity system.
Langerhans cells are paradigmatic dendriticcells, described in 1868 by a youngmedical student, Paul Langerhans in Berlin. Langerhans cells are present withepithelial cells in the epidermis, bronchi and mucosae. After antigenic challenge,Langerhans cells migrate into the T cell areas of proximal lymph nodes where they actas professional antigen-presenting cells.
Transcutaneous immunization (TCI) is a novel strategy for administering adjuvantand antigen to the skin surface. The adjuvant and antigen applied using TCIapparently target Langerhans cells in the skin, eliciting systemic antibodies, includingrobust mucosal IgG and secretory IgA responses in both mice and humans andprotective immunity against mucosal challenge with toxin or live virus.
Karande describe particular mixtures of penetration enhancers that increase skinpermeability to macromolecules (＜1-10 kDa) by up to＜100-fold without inducingskin irritation. In vitro experiments demonstrated that the mixtures deliveredmacromolecular drugs, including heparin, leutinizing hormone releasing hormone(LHRH) and oligonulceotides across the skin. In vivo experiments on hairless ratswith leuprolide acetate confirmed the potency and safety of one such mixture, sodiumlaureth sulfate (SLA) and phenylpiperazine (PP).
Imiquimod, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (also knownas R-837 and S-26308), is a nonnucleoside heterocyclic amine that belongs to a classof products known as immune-response modifiers. In vivo studies have demonstratedthat imiquimod is a potent inducer of alpha IFN(IFN-a), tumor necrosis factor alpha(TNF-a), and interleukin-1,6,8,10,12 (IL-1,6,8,10,12) and that it has adjuvantproperties greater than those of Freund's complete adjuvant. In animal models,imiquimod has been demonstrated to have potent antiviral and antitumor effects.
We use the imiquimod as adjuvant to investigate the adjuvantal effect of imiquimod on transcutaneous immunization for subunit antigen, virus antigen and DNA vaccine.
SLA: PP (weight fractions,0.7:0.3;total concentration of 0.5%(wt/vol)) dissolves in1:1 phosphate buffered saline(PBS):ethanol. This solution admixes with HBV subunitantigen, HAV antigen and HBsAg DNA vaccine distinctively. The admixtures werebesmeared on the depilated mouse back and then the adjuvant imiquimod wasbesmeared on the back. At 2, 4, 6, 8, 12, 20 weeks post inoculation. The specificHBsAg antibody were detected early at two weeks after transcutaneous immunization.The titers reached the highest level at 8 weeks. The array without imiquimod coulddetect antibody only at 4 weeks post immunization.
Conclusion The adjuvant imiquimod can enhance specific antibody levelsignificantly and quickly for HBsAg and HBsAg DNA vaccine.
引文
[1] Streilein JW. Skin-associated lymphoid tissues (SALT): origins and functions. J Invest Dermatol. 1983 Jun;80 Suppl:12s-16s.
[2] Bos JD, Kap senberg ML; The skin immune system (SIS):its cellular constituents and their interaction. [J] Immunol Today.1986;7(6):235
[3] Nicko off BJ; The immunology of dermis in dermal immune system [J] Boca Raton :CRC press 1993
[4] Schmitt D; The Langerhans cell: from in vitro production to use in cellular immunotherapy J Soc Biol. 2001;195(1):69-74
[5] Romani N ,Ratzinger G, Pfaller K, et al. [J]. Int Rev Cytol ,2001,207:237-270.
[6] 骆丹 缪旭 吉玺 等，人表皮朗格汉斯细胞的分离纯化及其对咪喹莫特免疫刺激作用的反应；中国免疫学杂志2007年第23卷147—150
[7] Glenn, G.M., Scharton-Kersten, T., Vassell, R. et al. Transcutaneous immunization with bacterial ADP-ribosylating exotoxins as antigens and adjuvants. Infect. Immun. 1999, 67:1100-1106.
[8] Glenn, G.M., et .al. Transcutaneous immunization with cholera toxin protects mice against lethal mucosal toxin challenge. J. Immunol. 1998.161:3211-3214.
[9] Berry, L.J., et al. Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection. Infect. Immun. 2004. 72:1019-1028.
[10] Yu, J., et al. Transcutaneous immunization using colonization factor and heat-labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli. Infect. Immun. 2002, 70:1056-1068.
[11] Guerena-Burgueno, F.et al. Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously. Infect. Immun. 2002.70:1874-1880.
[12] Guebre-Xabier, M., et al. Immunostimulant patch containing heat-labile enterotoxin from Escherichia coli enhances immune responses to injected influenza virus vaccine through activation of skin dendritic cells. J. Virol. 2003.77:5218-5225.
[13] Godefroy, S., et al. Immunization onto shaved skin with a bacterial enterotoxin adjuvant protects mice against respiratory syncytial virus (RSV). Vaccine. 2003.21:1665-1671.
[14] Seo, N., et al. Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis. Proc.Natl. Acad. Sci. U. S. A. 2000.97:371-376.
[15] Kahlon, R., et al. Optimization of epicutaneous immunization for the induction of CTL.Vaccine. 2003.21:2890-2899.
[16] Hammond, S.A., Walwender, D., Alving, C.R. et al Transcutaneous immunization:T cell responses and boosting of existing immunity. Vaccine. 2001. 19:2701-2707.
[17] Baca-Estrada, M.E., et al. The haemopoietic growth factor, Flt3L, alters the immune response induced by transcutaneous immunization. Immunology. 2002.107:69-76.
[18] A.-S. Beignon, J.-P. Briand, S. Muller et al.Immunization onto bare skin with heat-labile enterotoxin of Escherichia coli enhances immune responses to coadministered protein and peptide antigens and protects mice against lethal toxin challenge. Immunology 2001.102:344-351
[19] Kristina K. Peachman,l Mangala Rao,l Carl R. Alving, et al. Correlation between Lethal Toxin-Neutralizing Antibody Titers and Protection from Intranasal Challenge with Bacillus anthracis Ames Strain Spores in Mice after Transcutaneous Immunization with Recombinant Anthrax Protective Antigen .Infection And Immunity, Jan. 2006, Vol. 74, No. 1 ,p. 794-797
[20] Michniak B B , player M R , Godwin D A, et al. A study of enhancer structure activity relationships in a series of 2-oxopiperidine-1-acetic acid esters[J ]. Research, 1995 ,13 (9) :268.
[21] Karande P, Jain A, Mitragotri S; Discovery of transdermal penetration enhancers by high-throughput screening [J] Nat Biotechnol. 2004 Feb;22(2): 192-7
[22] Williams NA, Hirst TR, Nashar TO. Immune modulation by the cholera O like enterotoxins :from adjuvant to therapeutic. Immunol Today ,1999 ,20(2) :95
[23] Nashar TO, Williams NA, Hirst TR. Importance of receptor binding in the immunogenicity, adjuvanticity and therapeutic properties of cholera toxin and Escherichia coli heatOlabile enterotoxin. Med Microbiol Immunol ,1998 1187(1) :3
[24] Lencer WI, Hirst TR, Holmes RK..Membrane traffic and the cellular uptake of cholera toxin. Biochim BiophysActa, 1999, 8; 1450(3) :177
[25] Hovey BT, Verlinde CL, Merritt EA et al. StructureObased discovery of a poreObinding ligand : towards assembly inhibitors for cholera and related AB5 toxins. J Mol Biol,1999,285(3) :1169
[26] Cheng E, CardenasOFreytagL, Clements JD. The role of cAMP in mucosal adjuvanticity of Escherichia coli heatOlabile enterotoxin (LT). Vaccine ,1999,18 (102) :38
[27] Rappuoli R, Pizza M, Douce Get al. Structure and mucosal adjuvanticity of cholera and Escherichia coli heatOlabile enterotoxins. Immunol Today, 1999, 20(11) :493
[28] Lycke N. The mechanism of cholera toxin adjuvanticity. Res Immunol,1997 ,148 (8O9):504
[29] De Haan L, Holtrop M, Verweij WR et al. Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coIi heatOlabile enterotoxin. Immunology ,1999,97(4) :706
[30] Karl R. Beutner, Stephen K. Tyring, Kenneth F et al .Imiquimod, a Patient-Applied Immune-Response Modifier for Treatment of External Genital Warts Antimicrobial Agents And Chemotherapy, 1998 ,Vol. 42, No. 4, p. 789-794
[31] 张敏，王京燕，王洪全，抗病毒新药—咪喹莫特的毒性实验研究，中国艾滋病性病 2004 年2月第10卷第1期，p31-32
[32] Kono, T., S. Kondo, S. Pastore, et al. Effects of a novel topical immunomodulator, imiquimod,on keratinocyte cytokine gene expression. Lymphokine Cytokine,1994,Res. 13:71-76.
[33] Miller, R. L., L. M. Imbertson, M. J. Reiter, et al. Inhibition of herpes simplex virus infectionsin a guinea pig model by S-26308, 1985 In Program and abstractsof the 25th Interscience Conference on Antimicrobial Agents and Chemotherapy.American Society for Microbiology, Washington, D.C. p. 235.
[34] Reiter, M J., T. L. Testerman, R. L. Miller, et al. Cytokine induction in mice by the immunomodulator imiquimod. J. Leukocyte Biol.1994.55:234-240.
[35]Stanley MA; Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential. [J] Clin Exp Dermatol 2002;27:571-577.
[36]Wagner T. L., Ahonen C. L., Couture A. M., et al; Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod. [J] Cell. Immunol. 1999;191:10-19
[37] Sidky, Y. A., C. E. Weeks, J. Hatcher, et al.Effects of treatment with the oral interferon inducer, R-837, on the growth of mouse colon carcinoma,. In Proceedings of the American Association for Cancer Research, 1990. MC-26, abstr. 2574, p. 433
[38] Takeda K, Kaisho T, A kira S: Toll-like receptors. Annu Rev Immunol, 2003,21: 335-376.
[39] Jarrossay D, Napolitani G, Colonna M, et al; Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. [J] Eur. J. Immunol. 2001;31:3388-93
[40]Ito T, Amakawa R, Kaisho T,et al: Interferon-aand interleukin-12 are induced differentially by Toll-like receptor 7 ligands in human blood dendritic cell subsets [J]. J. Exp. Med. 2002;195:1507-12
[41]Karen Honey; TLR ligands from the natural world ; [J] Nat Rev immunol 2004;4:247
[42] Jurk M, Heil F, Vollmer J et al.;Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. [J] Nat Immunol. 2002 Jun;3(6):499.
[43] Shizuo Akira, kiyoshi takeda et al.; Toll-like receptors: critical proteins linking innate and acquired immunity [J] Nat immunol ,August 2001,Volume 2: 675-680
[44] Hemmi H, Kaisho T, Takeuchi O et al.; Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. [J] Nat Immunol. 2002 Feb;3(2):196-200
[45] Burns RP Jr , Ferbel B , Tomai M, et al. The imidazoquinolines , imiquimod and R-848, induce functional, but not phenotypic, maturation of human epidermal Langerhans cells. [J] Clin Immunol, 2000,94 (1) :13-23.
[46] Tomai M. A., L. M. Imbertson, T. L. Stanczak, et al.; The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes.. [J] Cell. Immunol. 2000, 203:55.
[47] Gerd Rechtsteiner, Tobias Warger, Philipp Osterloh, et al; Priming of CTL by Transcutaneous Peptide Immunization with Imiquimod [J] J Immunol 2005,174:2476-2480
[48] 李义方，严玉兰，张树林，等．HBsAg阳性各类肝炎及携带者HBV复制指标及传染性的观察[J]中国流行病学杂志，1991，11(6)：388．
[49] 韩雅儒，王淑珍，宫荫乡，等．乙肝重组疫苗(痘苗表达)的研制与试用[J]．中华微生物学和免疫学杂志，1989，9
[50] Dean Johnston, Jean-Claude Bystryn: Imiquimod Is A Potent Adjuvant To A Weakly-Immunogenic Protein Prototype Vaccine. 2006, Vaccine, Volume 24, Issue 11, Pages 1958-1965
[51] Johnston D, Zaidi B, Bystryn JC ;TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines.Cancer Immunol Immunother. 2007 Aug;56(8):1133-41.
[52] Cheng WF, Hung CH, Chai CY, et al. Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen. J Virol, 2001,75(5):2368-2376.
[53] Hung CF, Cheng WF, Chai CY, et al. Improving vaccine potency through intercellular spreading and enhanced MHC class Ⅰ presentation of antigen. J Immunol, 20011166(9):5733-5740.
[54] Michel N, Osen W, Gissmann L, et al. Enhanced immunogenicity of HPV 16 E7 fusion proteins in DNAvaccination. Virology, 2002,294(1):47-59.
[55] Kim TW, Hung CF, Kim JW, et al. Vaccination with a DNA vaccine encoding herpes simplex virus type 1 VP22 linked to antigen generates long-term antigen-specific CD8-positive memory T cells and protective immunity. Hum Gene Ther,2004,15(2): 167-177.
[56] Hung CF, He L, Juang J,et al. Improving DNA vaccine potency by linking Marek's disease virus type 1 VP22 to an antigen. J Virol, 2002,76(6): 2676-2682.
[57] ZUBERAnne Kjerrstrom ; BRAVE Andreas ; ENGSTROM Gunnel ; et al. Topical delivery of imiquimod to a mouse model as a novel adjuvant for human immunodeficiency virus (HIV) DNA .Vaccine ,2004, vol. 22, p.1791-1798
[58] Lindy L. Thomsen, Peter Topley, Maria G. Daly et al.imiquimod and Resquimod in a mouse model: adjuvants for DNA vaccination by particle-mediated immunotherapeutic delivery. Vaccine,2004,vol.22,p.1799-1809
[59] LongPing Wen, Yongping Chen, Yuanyuan Shen et al. Transdermal protein delivery by a coadministered peptide identified via phage display. Nature Biotechnology 2006,24, 455-460
[60] 黄胜炎．无针头注射剂的开发近况[J]。国外医药一合成药、生化药、制剂分册，2000，20(1)：48-49．
[1] Streilein JW; Skin-associated lymphoid tissues (SALT): origins and functions.[J] .J Invest Dermatol. 1983 Jun;80 Supph12s-16s
[2] Bos JD, Kap senberg ML; The skin immune system (SIS):its cellular constituents and their interaction. [J] Immunol Today.1986;7(6):235
[3] Nickoloff BJ The immunology of dermis in dermal immune system [J] Boca Raton :CRC press 1993
[4] Karande P, Jain A, Mitragotri S; Discovery of transdermal penetration enhancers by high-throughput screening [J] Nat Biotechnol. 2004 Feb;22(2):192-7. Epub 2004 Jan 4
[5] 美国 FDA : Aldara
[6] Stanley MA; Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential. [J] Clin Exp Dermatol 2002;27:571-577.
[7] Wagner T. L., Ahonen C. L., Couture A. M., et al; Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod. [J] Cell. Immunol. 1999;191:10-19.
[8] Jarrossay D, Napolitani G, Colonna M, et al; Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells.[J] Eur. J. Immunoh 2001;31:3388-93
[9] Ito T, Amakawa R, Kaisho T,et al; Interferon-α and interleukin-12 are induced differentially by Toll-like receptor 7 ligands in human blood dendritic cell subsets[J]. J. Exp. Med. 2002;195:1507-12
[10] Karen Honey; TLR ligands from the natural world ; [J] Nat Rev immunol 2004;4:247
[11]Jurk M, Heil F, Vollmer J et al.;Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. [J] Nat Immunol. 2002 Jun;3(6):499.
[12] Shizuo Akira, kiyoshi takeda et al.; Toll-like receptors: critical proteins linking innate and acquired immunity [J] Nat immunol ,August 2001,Volume 2: 675-680
[13] Hemmi H, Kaisho T, Takeuchi 0 et al.; Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. [J] Nat Immunol. 2002 Feb;3(2):196-200
[14]Burns RP Jr, Ferbel B , Tomai M, et al. The imidazoquinolines , imiquimod and R - 848 , induce functional, but not phenotypic, maturation of human epidermal Langerhans cells. [J] Clin Immunol, 2000, 94 (1):13-23.
[15] Gail A. Bishop, Yina Hsing, Bruce S. Hostager, et al; Molecular Mechanisms of B Lymphocyte Activation by the Immune Response Modifier R-848, [J] J Immunol, 2000,165:5552-5557.
[16]Tomai M. A., L. M. Imbertson, T. L. Stanczak, et al.; The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes.. [J] Cell. Immunol. 2000, 203:55.
[17] Gerd Rechtsteiner, Tobias Warger, Philipp Osterloh, et al; Priming of CTL by Transcutaneous Peptide Immunization with Imiquimod [J] J Immunol 2005,174:2476-2480
[18]Babiuk S, Baca-Estrada M, Babiuk LA, et al. Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery; [J] J Controlled Release, 2000, 66(2-3): 199-214.
[2] Bos JD, Kap senberg ML; The skin immune system (SIS):its cellular constituents and their interaction. [J] Immunol Today.1986;7(6):235
[3] Nicko off BJ; The immunology of dermis in dermal immune system [J] Boca Raton :CRC press 1993
[4] Schmitt D; The Langerhans cell: from in vitro production to use in cellular immunotherapy J Soc Biol. 2001;195(1):69-74
[5] Romani N ,Ratzinger G, Pfaller K, et al. [J]. Int Rev Cytol ,2001,207:237-270.
[6] 骆丹 缪旭 吉玺 等，人表皮朗格汉斯细胞的分离纯化及其对咪喹莫特免疫刺激作用的反应；中国免疫学杂志2007年第23卷147—150
[7] Glenn, G.M., Scharton-Kersten, T., Vassell, R. et al. Transcutaneous immunization with bacterial ADP-ribosylating exotoxins as antigens and adjuvants. Infect. Immun. 1999, 67:1100-1106.
[8] Glenn, G.M., et .al. Transcutaneous immunization with cholera toxin protects mice against lethal mucosal toxin challenge. J. Immunol. 1998.161:3211-3214.
[9] Berry, L.J., et al. Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection. Infect. Immun. 2004. 72:1019-1028.
[10] Yu, J., et al. Transcutaneous immunization using colonization factor and heat-labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli. Infect. Immun. 2002, 70:1056-1068.
[11] Guerena-Burgueno, F.et al. Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously. Infect. Immun. 2002.70:1874-1880.
[12] Guebre-Xabier, M., et al. Immunostimulant patch containing heat-labile enterotoxin from Escherichia coli enhances immune responses to injected influenza virus vaccine through activation of skin dendritic cells. J. Virol. 2003.77:5218-5225.
[13] Godefroy, S., et al. Immunization onto shaved skin with a bacterial enterotoxin adjuvant protects mice against respiratory syncytial virus (RSV). Vaccine. 2003.21:1665-1671.
[14] Seo, N., et al. Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis. Proc.Natl. Acad. Sci. U. S. A. 2000.97:371-376.
[15] Kahlon, R., et al. Optimization of epicutaneous immunization for the induction of CTL.Vaccine. 2003.21:2890-2899.
[16] Hammond, S.A., Walwender, D., Alving, C.R. et al Transcutaneous immunization:T cell responses and boosting of existing immunity. Vaccine. 2001. 19:2701-2707.
[17] Baca-Estrada, M.E., et al. The haemopoietic growth factor, Flt3L, alters the immune response induced by transcutaneous immunization. Immunology. 2002.107:69-76.
[18] A.-S. Beignon, J.-P. Briand, S. Muller et al.Immunization onto bare skin with heat-labile enterotoxin of Escherichia coli enhances immune responses to coadministered protein and peptide antigens and protects mice against lethal toxin challenge. Immunology 2001.102:344-351
[19] Kristina K. Peachman,l Mangala Rao,l Carl R. Alving, et al. Correlation between Lethal Toxin-Neutralizing Antibody Titers and Protection from Intranasal Challenge with Bacillus anthracis Ames Strain Spores in Mice after Transcutaneous Immunization with Recombinant Anthrax Protective Antigen .Infection And Immunity, Jan. 2006, Vol. 74, No. 1 ,p. 794-797
[20] Michniak B B , player M R , Godwin D A, et al. A study of enhancer structure activity relationships in a series of 2-oxopiperidine-1-acetic acid esters[J ]. Research, 1995 ,13 (9) :268.
[21] Karande P, Jain A, Mitragotri S; Discovery of transdermal penetration enhancers by high-throughput screening [J] Nat Biotechnol. 2004 Feb;22(2): 192-7
[22] Williams NA, Hirst TR, Nashar TO. Immune modulation by the cholera O like enterotoxins :from adjuvant to therapeutic. Immunol Today ,1999 ,20(2) :95
[23] Nashar TO, Williams NA, Hirst TR. Importance of receptor binding in the immunogenicity, adjuvanticity and therapeutic properties of cholera toxin and Escherichia coli heatOlabile enterotoxin. Med Microbiol Immunol ,1998 1187(1) :3
[24] Lencer WI, Hirst TR, Holmes RK..Membrane traffic and the cellular uptake of cholera toxin. Biochim BiophysActa, 1999, 8; 1450(3) :177
[25] Hovey BT, Verlinde CL, Merritt EA et al. StructureObased discovery of a poreObinding ligand : towards assembly inhibitors for cholera and related AB5 toxins. J Mol Biol,1999,285(3) :1169
[26] Cheng E, CardenasOFreytagL, Clements JD. The role of cAMP in mucosal adjuvanticity of Escherichia coli heatOlabile enterotoxin (LT). Vaccine ,1999,18 (102) :38
[27] Rappuoli R, Pizza M, Douce Get al. Structure and mucosal adjuvanticity of cholera and Escherichia coli heatOlabile enterotoxins. Immunol Today, 1999, 20(11) :493
[28] Lycke N. The mechanism of cholera toxin adjuvanticity. Res Immunol,1997 ,148 (8O9):504
[29] De Haan L, Holtrop M, Verweij WR et al. Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coIi heatOlabile enterotoxin. Immunology ,1999,97(4) :706
[30] Karl R. Beutner, Stephen K. Tyring, Kenneth F et al .Imiquimod, a Patient-Applied Immune-Response Modifier for Treatment of External Genital Warts Antimicrobial Agents And Chemotherapy, 1998 ,Vol. 42, No. 4, p. 789-794
[31] 张敏，王京燕，王洪全，抗病毒新药—咪喹莫特的毒性实验研究，中国艾滋病性病 2004 年2月第10卷第1期，p31-32
[32] Kono, T., S. Kondo, S. Pastore, et al. Effects of a novel topical immunomodulator, imiquimod,on keratinocyte cytokine gene expression. Lymphokine Cytokine,1994,Res. 13:71-76.
[33] Miller, R. L., L. M. Imbertson, M. J. Reiter, et al. Inhibition of herpes simplex virus infectionsin a guinea pig model by S-26308, 1985 In Program and abstractsof the 25th Interscience Conference on Antimicrobial Agents and Chemotherapy.American Society for Microbiology, Washington, D.C. p. 235.
[34] Reiter, M J., T. L. Testerman, R. L. Miller, et al. Cytokine induction in mice by the immunomodulator imiquimod. J. Leukocyte Biol.1994.55:234-240.
[35]Stanley MA; Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential. [J] Clin Exp Dermatol 2002;27:571-577.
[36]Wagner T. L., Ahonen C. L., Couture A. M., et al; Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod. [J] Cell. Immunol. 1999;191:10-19
[37] Sidky, Y. A., C. E. Weeks, J. Hatcher, et al.Effects of treatment with the oral interferon inducer, R-837, on the growth of mouse colon carcinoma,. In Proceedings of the American Association for Cancer Research, 1990. MC-26, abstr. 2574, p. 433
[38] Takeda K, Kaisho T, A kira S: Toll-like receptors. Annu Rev Immunol, 2003,21: 335-376.
[39] Jarrossay D, Napolitani G, Colonna M, et al; Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. [J] Eur. J. Immunol. 2001;31:3388-93
[40]Ito T, Amakawa R, Kaisho T,et al: Interferon-aand interleukin-12 are induced differentially by Toll-like receptor 7 ligands in human blood dendritic cell subsets [J]. J. Exp. Med. 2002;195:1507-12
[41]Karen Honey; TLR ligands from the natural world ; [J] Nat Rev immunol 2004;4:247
[42] Jurk M, Heil F, Vollmer J et al.;Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. [J] Nat Immunol. 2002 Jun;3(6):499.
[43] Shizuo Akira, kiyoshi takeda et al.; Toll-like receptors: critical proteins linking innate and acquired immunity [J] Nat immunol ,August 2001,Volume 2: 675-680
[44] Hemmi H, Kaisho T, Takeuchi O et al.; Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. [J] Nat Immunol. 2002 Feb;3(2):196-200
[45] Burns RP Jr , Ferbel B , Tomai M, et al. The imidazoquinolines , imiquimod and R-848, induce functional, but not phenotypic, maturation of human epidermal Langerhans cells. [J] Clin Immunol, 2000,94 (1) :13-23.
[46] Tomai M. A., L. M. Imbertson, T. L. Stanczak, et al.; The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes.. [J] Cell. Immunol. 2000, 203:55.
[47] Gerd Rechtsteiner, Tobias Warger, Philipp Osterloh, et al; Priming of CTL by Transcutaneous Peptide Immunization with Imiquimod [J] J Immunol 2005,174:2476-2480
[48] 李义方，严玉兰，张树林，等．HBsAg阳性各类肝炎及携带者HBV复制指标及传染性的观察[J]中国流行病学杂志，1991，11(6)：388．
[49] 韩雅儒，王淑珍，宫荫乡，等．乙肝重组疫苗(痘苗表达)的研制与试用[J]．中华微生物学和免疫学杂志，1989，9
[50] Dean Johnston, Jean-Claude Bystryn: Imiquimod Is A Potent Adjuvant To A Weakly-Immunogenic Protein Prototype Vaccine. 2006, Vaccine, Volume 24, Issue 11, Pages 1958-1965
[51] Johnston D, Zaidi B, Bystryn JC ;TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines.Cancer Immunol Immunother. 2007 Aug;56(8):1133-41.
[52] Cheng WF, Hung CH, Chai CY, et al. Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen. J Virol, 2001,75(5):2368-2376.
[53] Hung CF, Cheng WF, Chai CY, et al. Improving vaccine potency through intercellular spreading and enhanced MHC class Ⅰ presentation of antigen. J Immunol, 20011166(9):5733-5740.
[54] Michel N, Osen W, Gissmann L, et al. Enhanced immunogenicity of HPV 16 E7 fusion proteins in DNAvaccination. Virology, 2002,294(1):47-59.
[55] Kim TW, Hung CF, Kim JW, et al. Vaccination with a DNA vaccine encoding herpes simplex virus type 1 VP22 linked to antigen generates long-term antigen-specific CD8-positive memory T cells and protective immunity. Hum Gene Ther,2004,15(2): 167-177.
[56] Hung CF, He L, Juang J,et al. Improving DNA vaccine potency by linking Marek's disease virus type 1 VP22 to an antigen. J Virol, 2002,76(6): 2676-2682.
[57] ZUBERAnne Kjerrstrom ; BRAVE Andreas ; ENGSTROM Gunnel ; et al. Topical delivery of imiquimod to a mouse model as a novel adjuvant for human immunodeficiency virus (HIV) DNA .Vaccine ,2004, vol. 22, p.1791-1798
[58] Lindy L. Thomsen, Peter Topley, Maria G. Daly et al.imiquimod and Resquimod in a mouse model: adjuvants for DNA vaccination by particle-mediated immunotherapeutic delivery. Vaccine,2004,vol.22,p.1799-1809
[59] LongPing Wen, Yongping Chen, Yuanyuan Shen et al. Transdermal protein delivery by a coadministered peptide identified via phage display. Nature Biotechnology 2006,24, 455-460
[60] 黄胜炎．无针头注射剂的开发近况[J]。国外医药一合成药、生化药、制剂分册，2000，20(1)：48-49．
[1] Streilein JW; Skin-associated lymphoid tissues (SALT): origins and functions.[J] .J Invest Dermatol. 1983 Jun;80 Supph12s-16s
[2] Bos JD, Kap senberg ML; The skin immune system (SIS):its cellular constituents and their interaction. [J] Immunol Today.1986;7(6):235
[3] Nickoloff BJ The immunology of dermis in dermal immune system [J] Boca Raton :CRC press 1993
[4] Karande P, Jain A, Mitragotri S; Discovery of transdermal penetration enhancers by high-throughput screening [J] Nat Biotechnol. 2004 Feb;22(2):192-7. Epub 2004 Jan 4
[5] 美国 FDA : Aldara
[6] Stanley MA; Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential. [J] Clin Exp Dermatol 2002;27:571-577.
[7] Wagner T. L., Ahonen C. L., Couture A. M., et al; Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod. [J] Cell. Immunol. 1999;191:10-19.
[8] Jarrossay D, Napolitani G, Colonna M, et al; Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells.[J] Eur. J. Immunoh 2001;31:3388-93
[9] Ito T, Amakawa R, Kaisho T,et al; Interferon-α and interleukin-12 are induced differentially by Toll-like receptor 7 ligands in human blood dendritic cell subsets[J]. J. Exp. Med. 2002;195:1507-12
[10] Karen Honey; TLR ligands from the natural world ; [J] Nat Rev immunol 2004;4:247
[11]Jurk M, Heil F, Vollmer J et al.;Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. [J] Nat Immunol. 2002 Jun;3(6):499.
[12] Shizuo Akira, kiyoshi takeda et al.; Toll-like receptors: critical proteins linking innate and acquired immunity [J] Nat immunol ,August 2001,Volume 2: 675-680
[13] Hemmi H, Kaisho T, Takeuchi 0 et al.; Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. [J] Nat Immunol. 2002 Feb;3(2):196-200
[14]Burns RP Jr, Ferbel B , Tomai M, et al. The imidazoquinolines , imiquimod and R - 848 , induce functional, but not phenotypic, maturation of human epidermal Langerhans cells. [J] Clin Immunol, 2000, 94 (1):13-23.
[15] Gail A. Bishop, Yina Hsing, Bruce S. Hostager, et al; Molecular Mechanisms of B Lymphocyte Activation by the Immune Response Modifier R-848, [J] J Immunol, 2000,165:5552-5557.
[16]Tomai M. A., L. M. Imbertson, T. L. Stanczak, et al.; The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes.. [J] Cell. Immunol. 2000, 203:55.
[17] Gerd Rechtsteiner, Tobias Warger, Philipp Osterloh, et al; Priming of CTL by Transcutaneous Peptide Immunization with Imiquimod [J] J Immunol 2005,174:2476-2480
[18]Babiuk S, Baca-Estrada M, Babiuk LA, et al. Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery; [J] J Controlled Release, 2000, 66(2-3): 199-214.