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Volumn 30, Issue 36, 2012, Pages 5417-5424

Human papillomavirus 16 L1-E7 chimeric virus like particles show prophylactic and therapeutic efficacy in murine model of cervical cancer

Author keywords

Chimeric vaccine; CVLP vaccine; HPV vaccine; HPV16 vaccine

Indexed keywords

ANTIVIRUS AGENT; NEUTRALIZING ANTIBODY; RECOMBINANT PROTEIN;

EID: 84863999429     PISSN: 0264410X     EISSN: 18732518     Source Type: Journal    
DOI: 10.1016/j.vaccine.2012.06.010     Document Type: Article
Times cited : (14)

References (42)
  • 1
    • 0036848616 scopus 로고    scopus 로고
    • The viral etiology of cervical cancer
    • Bosch F.X., Munoz N. The viral etiology of cervical cancer. Virus Res 2002, 89(2):183-190.
    • (2002) Virus Res , vol.89 , Issue.2 , pp. 183-190
    • Bosch, F.X.1    Munoz, N.2
  • 2
    • 0036559896 scopus 로고    scopus 로고
    • Papillomaviruses and cancer: from basic studies to clinical application
    • zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002, 2(5):342-350.
    • (2002) Nat Rev Cancer , vol.2 , Issue.5 , pp. 342-350
    • zur Hausen, H.1
  • 3
    • 33748761925 scopus 로고    scopus 로고
    • Chapter 2: the burden of HPV-related cancers
    • S3/11-S3/25
    • Parkin D.M., Bray F. Chapter 2: the burden of HPV-related cancers. Vaccine 2006, 24(Suppl. 3). S3/11-S3/25.
    • (2006) Vaccine , vol.24 , Issue.SUPPL. 3
    • Parkin, D.M.1    Bray, F.2
  • 4
    • 8644252813 scopus 로고    scopus 로고
    • Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX adjuvant in women with cervical intraepithelial neoplasia
    • Frazer I.H., Quinn M., Nicklin J.L., Tan J., Perrin L.C., Ng P., et al. Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX adjuvant in women with cervical intraepithelial neoplasia. Vaccine 2004, 23(2):172-181.
    • (2004) Vaccine , vol.23 , Issue.2 , pp. 172-181
    • Frazer, I.H.1    Quinn, M.2    Nicklin, J.L.3    Tan, J.4    Perrin, L.C.5    Ng, P.6
  • 5
    • 0027428414 scopus 로고
    • Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles
    • Kirnbauer R., Taub J., Greenstone H., Roden R., Durst M., Gissmann L., et al. Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles. J Virol 1993, 67(12):6929-6936.
    • (1993) J Virol , vol.67 , Issue.12 , pp. 6929-6936
    • Kirnbauer, R.1    Taub, J.2    Greenstone, H.3    Roden, R.4    Durst, M.5    Gissmann, L.6
  • 6
    • 0029610072 scopus 로고
    • Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas
    • Suzich J.A., Ghim S.J., Palmer-Hill F.J., White W.I., Tamura J.K., Bell J.A., et al. Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proc Natl Acad Sci USA 1995, 92(25):11553-11557.
    • (1995) Proc Natl Acad Sci USA , vol.92 , Issue.25 , pp. 11553-11557
    • Suzich, J.A.1    Ghim, S.J.2    Palmer-Hill, F.J.3    White, W.I.4    Tamura, J.K.5    Bell, J.A.6
  • 8
    • 0036511592 scopus 로고    scopus 로고
    • Human papillomavirus in cervical cancer
    • Bosch F.X., de Sanjose S. Human papillomavirus in cervical cancer. Curr Oncol Rep 2002, 4(2):175-183.
    • (2002) Curr Oncol Rep , vol.4 , Issue.2 , pp. 175-183
    • Bosch, F.X.1    de Sanjose, S.2
  • 9
    • 34248364284 scopus 로고    scopus 로고
    • Introducing HPV, vaccine in developing countries-key challenges and issues
    • Agosti J.M., Goldie S.J. Introducing HPV, vaccine in developing countries-key challenges and issues. N Engl J Med 2007, 356(19):1908-1910.
    • (2007) N Engl J Med , vol.356 , Issue.19 , pp. 1908-1910
    • Agosti, J.M.1    Goldie, S.J.2
  • 10
    • 0036849736 scopus 로고    scopus 로고
    • The status of HPV16-specific T-cell reactivity in health and disease as a guide to HPV vaccine development
    • van der Burg S.H., de Jong A., Welters M.J., Offringa R., Melief C.J. The status of HPV16-specific T-cell reactivity in health and disease as a guide to HPV vaccine development. Virus Res 2002, 89(2):275-284.
    • (2002) Virus Res , vol.89 , Issue.2 , pp. 275-284
    • van der Burg, S.H.1    de Jong, A.2    Welters, M.J.3    Offringa, R.4    Melief, C.J.5
  • 11
    • 0030934907 scopus 로고    scopus 로고
    • Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia
    • Nakagawa M., Stites D.P., Farhat S., Sisler J.R., Moss B., Kong F., et al. Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia. J Infect Dis 1997, 175(4):927-931.
    • (1997) J Infect Dis , vol.175 , Issue.4 , pp. 927-931
    • Nakagawa, M.1    Stites, D.P.2    Farhat, S.3    Sisler, J.R.4    Moss, B.5    Kong, F.6
  • 12
    • 0033044742 scopus 로고    scopus 로고
    • Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumor protection
    • Schafer K., Muller M., Faath S., Henn A., Osen W., Zentgraf H., et al. Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumor protection. Int J Cancer 1999, 81(6):881-888.
    • (1999) Int J Cancer , vol.81 , Issue.6 , pp. 881-888
    • Schafer, K.1    Muller, M.2    Faath, S.3    Henn, A.4    Osen, W.5    Zentgraf, H.6
  • 13
    • 0032484471 scopus 로고    scopus 로고
    • Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway
    • Peng S., Frazer I.H., Fernando G.J., Zhou J. Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway. Virology 1998, 240(1):147-157.
    • (1998) Virology , vol.240 , Issue.1 , pp. 147-157
    • Peng, S.1    Frazer, I.H.2    Fernando, G.J.3    Zhou, J.4
  • 14
    • 0032539564 scopus 로고    scopus 로고
    • Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model
    • Greenstone H.L., Nieland J.D., de Visser K.E., De Bruijn M.L., Kirnbauer R., Roden R.B., et al. Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model. Proc Natl Acad Sci USA 1998, 95(4):1800-1805.
    • (1998) Proc Natl Acad Sci USA , vol.95 , Issue.4 , pp. 1800-1805
    • Greenstone, H.L.1    Nieland, J.D.2    de Visser, K.E.3    De Bruijn, M.L.4    Kirnbauer, R.5    Roden, R.B.6
  • 15
    • 0034254994 scopus 로고    scopus 로고
    • Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes
    • Liu W.J., Liu X.S., Zhao K.N., Leggatt G.R., Frazer I.H. Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes. Virology 2000, 273(2):374-382.
    • (2000) Virology , vol.273 , Issue.2 , pp. 374-382
    • Liu, W.J.1    Liu, X.S.2    Zhao, K.N.3    Leggatt, G.R.4    Frazer, I.H.5
  • 16
    • 0036239831 scopus 로고    scopus 로고
    • Route of administration of chimeric BPV1 VLP determines the character of the induced immune responses
    • Liu X.S., Liu W.J., Zhao K.N., Liu Y.H., Leggatt G., Frazer I.H. Route of administration of chimeric BPV1 VLP determines the character of the induced immune responses. Immunol Cell Biol 2002, 80(1):21-29.
    • (2002) Immunol Cell Biol , vol.80 , Issue.1 , pp. 21-29
    • Liu, X.S.1    Liu, W.J.2    Zhao, K.N.3    Liu, Y.H.4    Leggatt, G.5    Frazer, I.H.6
  • 17
    • 0038107884 scopus 로고    scopus 로고
    • Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16
    • Varsani A., Williamson A.L., de Villiers D., Becker I., Christensen N.D., Rybicki E.P. Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16. J Virol 2003, 77(15):8386-8393.
    • (2003) J Virol , vol.77 , Issue.15 , pp. 8386-8393
    • Varsani, A.1    Williamson, A.L.2    de Villiers, D.3    Becker, I.4    Christensen, N.D.5    Rybicki, E.P.6
  • 19
    • 0035871993 scopus 로고    scopus 로고
    • HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination
    • Kaufmann A.M., Nieland J., Schinz M., Nonn M., Gabelsberger J., Meissner H., et al. HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination. Int J Cancer 2001, 92(2):285-293.
    • (2001) Int J Cancer , vol.92 , Issue.2 , pp. 285-293
    • Kaufmann, A.M.1    Nieland, J.2    Schinz, M.3    Nonn, M.4    Gabelsberger, J.5    Meissner, H.6
  • 21
    • 33750319866 scopus 로고    scopus 로고
    • A deletion and point mutation study of the human papillomavirus type 16 major capsid gene
    • Varsani A., Williamson A.L., Jaffer M.A., Rybicki E.P. A deletion and point mutation study of the human papillomavirus type 16 major capsid gene. Virus Res 2006, 122(1-2):154-163.
    • (2006) Virus Res , vol.122 , Issue.1-2 , pp. 154-163
    • Varsani, A.1    Williamson, A.L.2    Jaffer, M.A.3    Rybicki, E.P.4
  • 22
    • 66149134832 scopus 로고    scopus 로고
    • Molecular variants of HPV-16 associated with cervical cancer in Indian population
    • Pillai M.R., Hariharan R., Babu J.M., Lakshmi S., Chiplunkar S.V., Patkar M., et al. Molecular variants of HPV-16 associated with cervical cancer in Indian population. Int J Cancer 2009, 125(1):91-103.
    • (2009) Int J Cancer , vol.125 , Issue.1 , pp. 91-103
    • Pillai, M.R.1    Hariharan, R.2    Babu, J.M.3    Lakshmi, S.4    Chiplunkar, S.V.5    Patkar, M.6
  • 23
    • 49849092724 scopus 로고    scopus 로고
    • Human papillomavirus type 16 L1E7 chimeric capsomeres have prophylactic and therapeutic efficacy against papillomavirus in mice
    • Bian T., Wang Y., Lu Z., Ye Z., Zhao L., Ren J., et al. Human papillomavirus type 16 L1E7 chimeric capsomeres have prophylactic and therapeutic efficacy against papillomavirus in mice. Mol Cancer Ther 2008, 7(5):1329-1335.
    • (2008) Mol Cancer Ther , vol.7 , Issue.5 , pp. 1329-1335
    • Bian, T.1    Wang, Y.2    Lu, Z.3    Ye, Z.4    Zhao, L.5    Ren, J.6
  • 24
    • 0029012828 scopus 로고
    • Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor
    • Roden R.B., Hubbert N.L., Kirnbauer R., Breitburd F., Lowy D.R., Schiller J.T. Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor. J Virol 1995, 69(8):5147-5151.
    • (1995) J Virol , vol.69 , Issue.8 , pp. 5147-5151
    • Roden, R.B.1    Hubbert, N.L.2    Kirnbauer, R.3    Breitburd, F.4    Lowy, D.R.5    Schiller, J.T.6
  • 25
    • 0021061819 scopus 로고
    • Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
    • Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65(1-2):55-63.
    • (1983) J Immunol Methods , vol.65 , Issue.1-2 , pp. 55-63
    • Mosmann, T.1
  • 26
    • 0027320440 scopus 로고
    • Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells
    • Feltkamp M.C., Smits H.L., Vierboom M.P., Minnaar R.P., de Jongh B.M., Drijfhout J.W., et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol 1993, 23(9):2242-2249.
    • (1993) Eur J Immunol , vol.23 , Issue.9 , pp. 2242-2249
    • Feltkamp, M.C.1    Smits, H.L.2    Vierboom, M.P.3    Minnaar, R.P.4    de Jongh, B.M.5    Drijfhout, J.W.6
  • 27
    • 0344406986 scopus 로고    scopus 로고
    • Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice
    • Ohlschlager P., Osen W., Dell K., Faath S., Garcea R.L., Jochmus I., et al. Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice. J Virol 2003, 77(8):4635-4645.
    • (2003) J Virol , vol.77 , Issue.8 , pp. 4635-4645
    • Ohlschlager, P.1    Osen, W.2    Dell, K.3    Faath, S.4    Garcea, R.L.5    Jochmus, I.6
  • 28
    • 0023738916 scopus 로고
    • A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity
    • Decker T., Lohmann-Matthes M.L. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods 1988, 115(1):61-69.
    • (1988) J Immunol Methods , vol.115 , Issue.1 , pp. 61-69
    • Decker, T.1    Lohmann-Matthes, M.L.2
  • 29
    • 70449338341 scopus 로고    scopus 로고
    • A pooled analysis of continued prophylactic efficacy of quadrivalent human papillomavirus (Types 6/11/16/18) vaccine against high-grade cervical and external genital lesions
    • Kjaer S.K., Sigurdsson K., Iversen O.E., Hernandez-Avila M., Wheeler C.M., Perez G., et al. A pooled analysis of continued prophylactic efficacy of quadrivalent human papillomavirus (Types 6/11/16/18) vaccine against high-grade cervical and external genital lesions. Cancer Prev Res (Phila) 2009, 2(10):868-878.
    • (2009) Cancer Prev Res (Phila) , vol.2 , Issue.10 , pp. 868-878
    • Kjaer, S.K.1    Sigurdsson, K.2    Iversen, O.E.3    Hernandez-Avila, M.4    Wheeler, C.M.5    Perez, G.6
  • 30
    • 74049150705 scopus 로고    scopus 로고
    • Evaluation of quadrivalent HPV 6/11/16/18 vaccine efficacy against cervical and anogenital disease in subjects with serological evidence of prior vaccine type HPV infection
    • Olsson S.E., Kjaer S.K., Sigurdsson K., Iversen O.E., Hernandez-Avila M., Wheeler C.M., et al. Evaluation of quadrivalent HPV 6/11/16/18 vaccine efficacy against cervical and anogenital disease in subjects with serological evidence of prior vaccine type HPV infection. Hum Vaccine 2009, 5(10):696-704.
    • (2009) Hum Vaccine , vol.5 , Issue.10 , pp. 696-704
    • Olsson, S.E.1    Kjaer, S.K.2    Sigurdsson, K.3    Iversen, O.E.4    Hernandez-Avila, M.5    Wheeler, C.M.6
  • 31
    • 67651049056 scopus 로고    scopus 로고
    • 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
    • Paavonen J., Naud P., Salmeron J., Wheeler C.M., Chow S.N., Apter D., et al. 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 2009, 374(9686):301-314.
    • (2009) Lancet , vol.374 , Issue.9686 , pp. 301-314
    • Paavonen, J.1    Naud, P.2    Salmeron, J.3    Wheeler, C.M.4    Chow, S.N.5    Apter, D.6
  • 32
    • 36248945563 scopus 로고    scopus 로고
    • Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (CIN 2/3)
    • Kaufmann A.M., Nieland J.D., Jochmus I., Baur S., Friese K., Gabelsberger J., et al. Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (CIN 2/3). Int J Cancer 2007, 121(12):2794-2800.
    • (2007) Int J Cancer , vol.121 , Issue.12 , pp. 2794-2800
    • Kaufmann, A.M.1    Nieland, J.D.2    Jochmus, I.3    Baur, S.4    Friese, K.5    Gabelsberger, J.6
  • 33
    • 0033696899 scopus 로고    scopus 로고
    • Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16
    • Chen X.S., Garcea R.L., Goldberg I., Casini G., Harrison S.C. Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16. Mol Cell 2000, 5(3):557-567.
    • (2000) Mol Cell , vol.5 , Issue.3 , pp. 557-567
    • Chen, X.S.1    Garcea, R.L.2    Goldberg, I.3    Casini, G.4    Harrison, S.C.5
  • 34
    • 34247106805 scopus 로고    scopus 로고
    • Aluminum hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release
    • Li H., Nookala S., Re F. Aluminum hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release. J Immunol 2007, 178(8):5271-5276.
    • (2007) J Immunol , vol.178 , Issue.8 , pp. 5271-5276
    • Li, H.1    Nookala, S.2    Re, F.3
  • 35
    • 0034085544 scopus 로고    scopus 로고
    • Comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection
    • Carter J.J., Koutsky L.A., Hughes J.P., Lee S.K., Kuypers J., Kiviat N., et al. Comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection. J Infect Dis 2000, 181(6):1911-1919.
    • (2000) J Infect Dis , vol.181 , Issue.6 , pp. 1911-1919
    • Carter, J.J.1    Koutsky, L.A.2    Hughes, J.P.3    Lee, S.K.4    Kuypers, J.5    Kiviat, N.6
  • 36
    • 0030724380 scopus 로고    scopus 로고
    • Assessment of cytotoxic T-lymphocyte phenotype using the specific markers granzyme B and TIA-1 in cervical neoplastic lesions
    • Bontkes H.J., de Gruijl T.D., Walboomers J.M., van den Muysenberg A.J., Gunther A.W., Scheper R.J., et al. Assessment of cytotoxic T-lymphocyte phenotype using the specific markers granzyme B and TIA-1 in cervical neoplastic lesions. Br J Cancer 1997, 76(10):1353-1360.
    • (1997) Br J Cancer , vol.76 , Issue.10 , pp. 1353-1360
    • Bontkes, H.J.1    de Gruijl, T.D.2    Walboomers, J.M.3    van den Muysenberg, A.J.4    Gunther, A.W.5    Scheper, R.J.6
  • 37
    • 7944235613 scopus 로고    scopus 로고
    • Impaired antigen presentation and effectiveness of combined active/passive immunotherapy for epithelial tumors
    • Matsumoto K., Leggatt G.R., Zhong J., Liu X., de Kluyver R.L., Peters T., et al. Impaired antigen presentation and effectiveness of combined active/passive immunotherapy for epithelial tumors. J Natl Cancer Inst 2004, 96(21):1611-1619.
    • (2004) J Natl Cancer Inst , vol.96 , Issue.21 , pp. 1611-1619
    • Matsumoto, K.1    Leggatt, G.R.2    Zhong, J.3    Liu, X.4    de Kluyver, R.L.5    Peters, T.6
  • 39
    • 0034003977 scopus 로고    scopus 로고
    • Boosting with recombinant vaccinia increases HPV-16 E7-specific T cell precursor frequencies of HPV-16 E7-expressing DNA vaccines
    • Chen C.H., Wang T.L., Hung C.F., Pardoll D.M., Wu T.C. Boosting with recombinant vaccinia increases HPV-16 E7-specific T cell precursor frequencies of HPV-16 E7-expressing DNA vaccines. Vaccine 2000, 18(19):2015-2022.
    • (2000) Vaccine , vol.18 , Issue.19 , pp. 2015-2022
    • Chen, C.H.1    Wang, T.L.2    Hung, C.F.3    Pardoll, D.M.4    Wu, T.C.5
  • 41
    • 0032779757 scopus 로고    scopus 로고
    • CD4-positive and CD8-positive cytotoxic T lymphocytes contribute to human papillomavirus type 16 E6 and E7 responses
    • Nakagawa M., Stites D.P., Palefsky J.M., Kneass Z., Moscicki A.B. CD4-positive and CD8-positive cytotoxic T lymphocytes contribute to human papillomavirus type 16 E6 and E7 responses. Clin Diagn Lab Immunol 1999, 6(4):494-498.
    • (1999) Clin Diagn Lab Immunol , vol.6 , Issue.4 , pp. 494-498
    • Nakagawa, M.1    Stites, D.P.2    Palefsky, J.M.3    Kneass, Z.4    Moscicki, A.B.5
  • 42
    • 68949125247 scopus 로고    scopus 로고
    • IFN-gamma promotes generation of IL-10 secreting CD4+ T cells that suppress generation of CD8 responses in an antigen-experienced host
    • Liu X.S., Leerberg J., MacDonald K., Leggatt G.R., Frazer I.H. IFN-gamma promotes generation of IL-10 secreting CD4+ T cells that suppress generation of CD8 responses in an antigen-experienced host. J Immunol 2009, 183(1):51-58.
    • (2009) J Immunol , vol.183 , Issue.1 , pp. 51-58
    • Liu, X.S.1    Leerberg, J.2    MacDonald, K.3    Leggatt, G.R.4    Frazer, I.H.5


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