This study developed and validated a high-throughput human papillomavirus (HPV) serology method based on Luminex technology, using pseudovirions (PsVs) of eight mucosal HPV types (HPV-6, -11, -16, -18, -31, -45, -52 and -58) and two cutaneous HPV types (HPV-5 and -38) bound to heparin-coated beads. Analysis with neutralizing type-specific monoclonal antibodies against the included HPV types indicated the type specificity of the assay. Analysis of negative-control serum samples from 63 children and 71 middle-aged women with up to one lifetime sexual partner indicated high specificity. Positive-control serum samples from subjects with known HPV DNA status or clinical diagnosis found expected sensitivities for most of the HPV types in 219 European serum samples, but lower than expected in 124 samples from Africa. HPV-45 and -52 did not react as expected with the human serum samples. The PsV-Luminex method was used to determine the HPV-seropositivity-associated relative risk for future cervical cancer using 208 serum samples from a prospective study of 18 814 women followed for 23 years, analysed previously with standard HPV-16 ELISA. The PsV-Luminex method gave similar results to ELISA (=0.77). As expected, HPV seropositivities assayed using the PsV-Luminex method found an increased risk of cervical cancer for HPV-16 [odds ratio (OR)=7.7, 95 % confidence interval (CI)=2.6–23] and HPV-31 (OR=4.1, 95 % CI=1.6–10.8), non-significant tendencies for increased risk for other mucosal HPV types and no risk for the cutaneous HPV types. In summary, multiplexed HPV serology using mammalian-derived PsVs selected for native conformation by binding to heparin-coated beads was validated as a high-throughput HPV serological method for most of the analysed HPV types.


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  1. af Geijersstam, V., Eklund, C., Wang, Z., Sapp, M., Schiller, J. T., Dillner, J. & Dillner, L.(1999). A survey of seroprevalence of human papillomavirus types 16, 18 and 33 among children. Int J Cancer 80, 489–493.[CrossRef] [Google Scholar]
  2. Antonsson, A., Karanfilovska, S., Lindqvist, P. G. & Hansson, B. G.(2003). General acquisition of human papillomavirus infections of skin occurs in early infancy. J Clin Microbiol 41, 2509–2514.[CrossRef] [Google Scholar]
  3. Arbyn, M. & Dillner, J.(2007). Review of current knowledge on HPV vaccination: an appendix to the European Guidelines for Quality Assurance in cervical cancer screening. J Clin Virol 38, 189–197.[CrossRef] [Google Scholar]
  4. Buck, C. B., Pastrana, D. V., Lowy, D. R. & Schiller, J. T.(2004). Efficient intracellular assembly of papillomaviral vectors. J Virol 78, 751–757.[CrossRef] [Google Scholar]
  5. Buck, C. B., Pastrana, D. V., Lowy, D. R. & Schiller, J. T.(2005). Generation of HPV pseudovirions using transfection and their use in neutralization assays. Methods Mol Med 119, 445–462. [Google Scholar]
  6. Carter, J. J., Koutsky, L. A., Wipf, G. C., Christensen, N. D., Lee, S. K., Kuypers, J., Kiviat, N. & Galloway, D. A.(1996). The natural history of human papillomavirus type 16 capsid antibodies among a cohort of university women. J Infect Dis 174, 927–936.[CrossRef] [Google Scholar]
  7. Christensen, N. D., Dillner, J., Eklund, C., Carter, J. J., Wipf, G. C., Reed, C. A., Cladel, N. M. & Galloway, D. A.(1996a). Surface conformational and linear epitopes on HPV-16 and HPV-18 L1 virus-like particles as defined by monoclonal antibodies. Virology 223, 174–184.[CrossRef] [Google Scholar]
  8. Christensen, N. D., Reed, C. A., Cladel, N. M., Hall, K. & Leiserowitz, G. S.(1996b). Monoclonal antibodies to HPV-6 L1 virus-like particles identify conformational and linear neutralizing epitopes on HPV-11 in addition to type-specific epitopes on HPV-6. Virology 224, 477–486.[CrossRef] [Google Scholar]
  9. Christensen, N. D., Cladel, N. M., Reed, C. A., Budgeon, L. R., Embers, M. E., Skulsky, D. M., McClements, W. L., Ludmerer, S. W. & Jansen, K. U.(2001). Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types. Virology 291, 324–334.[CrossRef] [Google Scholar]
  10. Combita, A. L., Touzé, A., Bousarghin, L., Sizaret, P. Y., Muñoz, N. & Coursaget, P.(2001). Gene transfer using human papillomavirus pseudovirions varies according to virus genotype and requires cell surface heparan sulfate. FEMS Microbiol Lett 204, 183–188.[CrossRef] [Google Scholar]
  11. Dias, D., Van Doren, J., Schlottmann, S., Kelly, S., Puchalski, D., Ruiz, W., Boerckel, P., Kessler, J., Antonello, J. M. & other authors(2005). Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18. Clin Diagn Lab Immunol 12, 959–969. [Google Scholar]
  12. Dillner, J., Wiklund, F., Lenner, P., Eklund, C., Frederiksson-Shanazarian, V., Schiller, J. T., Hibma, M., Hallmans, G. & Stendahl, U.(1995). Antibodies against linear and conformational epitopes of human papillomavirus type 16 that independently associate with incident cervical cancer. Int J Cancer 60, 377–382. [Google Scholar]
  13. Dillner, J., Andersson-Ellstrom, A., Hagmar, B. & Schiller, J.(1999). High risk genital papillomavirus infections are not spread vertically. Rev Med Virol 9, 23–29.[CrossRef] [Google Scholar]
  14. Drobni, P., Mistry, N., McMillan, N. & Evander, M.(2003). Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry. Virology 310, 163–172.[CrossRef] [Google Scholar]
  15. Ferguson, M., Heath, A., Johnes, S., Pagliusi, S. & Dillner, J.(2006). Results of the first WHO international collaborative study on the standardization of the detection of antibodies to human papillomaviruses. Int J Cancer 118, 1508–1514.[CrossRef] [Google Scholar]
  16. Forslund, O., Antonsson, A., Edlund, K., van den Brule, A. J., Hansson, B. G., Meijer, C. J., Ryd, W., Rylander, E., Strand, A. & other authors(2002). Population-based type-specific prevalence of high-risk human papillomavirus infection in middle-aged Swedish women. J Med Virol 66, 535–541.[CrossRef] [Google Scholar]
  17. Forslund, O., Iftner, T., Andersson, K., Lindelof, B., Hradil, E., Nordin, P., Stenquist, B., Kirnbauer, R., Dillner, J. & de Villiers, E. M.(2007). Cutaneous human papillomaviruses found in sun-exposed skin: beta-papillomavirus species 2 predominates in squamous cell carcinoma. J Infect Dis 196, 876–883.[CrossRef] [Google Scholar]
  18. Giroglou, T., Florin, L., Schafer, F., Streeck, R. E. & Sapp, M.(2001). Human papillomavirus infection requires cell surface heparan sulfate. J Virol 75, 1565–1570.[CrossRef] [Google Scholar]
  19. Grabowska, K., Wang, X., Jacobsson, A. & Dillner, J.(2002). Evaluation of cost-precision ratios of different strategies for ELISA measurement of serum antibody levels. J Immunol Methods 271, 1–15.[CrossRef] [Google Scholar]
  20. Hagensee, M. E., Yaegashi, N. & Galloway, D. A.(1993). Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins. J Virol 67, 315–322. [Google Scholar]
  21. Hakama, M., Lehtinen, M., Knekt, P., Aromaa, A., Leinikki, P., Miettinen, A., Paavonen, J., Peto, R. & Teppo, L.(1993). Serum antibodies and subsequent cervical neoplasms: a prospective study with 12 years of follow-up. Am J Epidemiol 137, 166–170. [Google Scholar]
  22. Harper, D. M., Franco, E. L., Wheeler, C., Ferris, D. G., Jenkins, D., Schuind, A., Zahaf, T., Innis, B., Naud, P. & other authors(2004). Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 364, 1757–1765.[CrossRef] [Google Scholar]
  23. Ho, G. Y., Burk, R. D., Klein, S., Kadish, A. S., Chang, C. J., Palan, P., Basu, J., Tachezy, R., Lewis, R. & Romney, S.(1995). Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia. J Natl Cancer Inst 87, 1365–1371.[CrossRef] [Google Scholar]
  24. Joyce, J. G., Tung, J. S., Przysiecki, C. T., Cook, J. C., Lehman, E. D., Sands, J. A., Jansen, K. U. & Keller, P. M.(1999). The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes. J Biol Chem 274, 5810–5822.[CrossRef] [Google Scholar]
  25. Kirnbauer, R., Booy, F., Cheng, N., Lowy, D. R. & Schiller, J. T.(1992). Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci U S A 89, 12180–12184.[CrossRef] [Google Scholar]
  26. Kjellberg, L., Wang, Z., Wiklund, F., Edlund, K., Angstrom, T., Lenner, P., Sjoberg, I., Hallmans, G., Wallin, K. L. & other authors(1999). Sexual behaviour and papillomavirus exposure in cervical intraepithelial neoplasia: a population-based case–control study. J Gen Virol 80, 391–398. [Google Scholar]
  27. Koutsky, L. A., Ault, K. A., Wheeler, C. M., Brown, D. R., Barr, E., Alvarez, F. B., Chiacchierini, L. M. & Jansen, K. U.(2002). A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 347, 1645–1651.[CrossRef] [Google Scholar]
  28. Landis, J. R. & Koch, G. G.(1977). An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers. Biometrics 33, 363–374.[CrossRef] [Google Scholar]
  29. Le Cann, P., Coursaget, P., Iochmann, S. & Touze, A.(1994). Self-assembly of human papillomavirus type 16 capsids by expression of the L1 protein in insect cells. FEMS Microbiol Lett 117, 269–274.[CrossRef] [Google Scholar]
  30. Lehtinen, M., Dillner, J., Knekt, P., Luostarinen, T., Aromaa, A., Kirnbauer, R., Koskela, P., Paavonen, J., Peto, R. & other authors(1996). Serologically diagnosed infection with human papillomavirus type 16 and risk for subsequent development of cervical carcinoma: nested case–control study. BMJ 312, 537–539.[CrossRef] [Google Scholar]
  31. Marais, D. J., Sampson, C. C., Urban, M. I., Sitas, F. & Wiliamson, A. L.(2007). The seroprevalence of IgG antibodies to human papillomavirus (HPV) types HPV-16, HPV-18, and HPV-11 capsid-antigens in mothers and their children. J Med Virol 79, 1370–1374.[CrossRef] [Google Scholar]
  32. Michael, K. M., Waterboer, T., Sehr, P., Rother, A., Reidel, U., Boeing, H., Bravo, I. G., Schlehofer, J., Gartner, B. C. & Pawlita, M.(2008). Seroprevalence of 34 human papillomavirus types in the German general population. PLoS Pathog 4, e1000091[CrossRef] [Google Scholar]
  33. Naucler, P., Da Costa, F. M., Ljungberg, O., Bugalho, A. & Dillner, J.(2004). Human papillomavirus genotypes in cervical cancers in Mozambique. J Gen Virol 85, 2189–2190.[CrossRef] [Google Scholar]
  34. Opalka, D., Lachman, C. E., MacMullen, S. A., Jansen, K. U., Smith, J. F., Chirmule, N. & Esser, M. T.(2003). Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay. Clin Diagn Lab Immunol 10, 108–115. [Google Scholar]
  35. Pagliusi, S. R., Dillner, J., Pawlita, M., Quint, W. G. V., Wheeler, C. M. & Ferguson, M.(2006). Chapter 23: International standard reagents for harmonization of HPV serology and DNA assays – an update. Vaccine 24 (Suppl. 3), S193–S200.[CrossRef] [Google Scholar]
  36. Pastrana, D. V., Buck, C. B., Pang, Y. Y., Thompson, C. D., Castle, P. E., FitzGerald, P. C., Kruger Kjaer, S., Lowy, D. R. & Schiller, J. T.(2004). Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18. Virology 321, 205–216.[CrossRef] [Google Scholar]
  37. Rizk, R. Z., Christensen, N. D., Michael, K. M., Muller, M., Sehr, P., Waterboer, T. & Pawlita, M.(2008). Reactivity pattern of 92 monoclonal antibodies with 15 human papillomavirus types. J Gen Virol 89, 117–129.[CrossRef] [Google Scholar]
  38. Rommel, O., Dillner, J., Fligge, C., Bergsdorf, C., Wang, X., Selinka, H. C. & Sapp, M.(2005). Heparan sulfate proteoglycans interact exclusively with conformationally intact HPV L1 assemblies: basis for a virus-like particle ELISA. J Med Virol 75, 114–121.[CrossRef] [Google Scholar]
  39. Sasagawa, T., Pushko, P., Steers, G., Gschmeissner, S. E., Hajibagheri, M. A., Finch, J., Crawford, L. & Tommasino, M.(1995). Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast Schizosaccharomyces pombe. Virology 206, 126–135.[CrossRef] [Google Scholar]
  40. Schiffman, M. H., Bauer, H. M., Hoover, R. N., Glass, A. G., Cadell, D. M., Rush, B. B., Scott, D. R., Sherman, M. E., Kurman, R. J. & other authors(1993). Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst 85, 958–964.[CrossRef] [Google Scholar]
  41. Shafti-Keramat, S., Handisurya, A., Kriehuber, E., Meneguzzi, G., Slupetzky, K. & Kirnbauer, R.(2003). Different heparan sulfate proteoglycans serve as cellular receptors for human papillomaviruses. J Virol 77, 13125–13135.[CrossRef] [Google Scholar]
  42. Silins, I., Wang, X., Tadesse, A., Jansen, K. U., Schiller, J. T., Avall-Lundqvist, E., Frankendal, B. & Dillner, J.(2004). A population-based study of cervical carcinoma and HPV infection in Latvia. Gynecol Oncol 93, 484–492.[CrossRef] [Google Scholar]
  43. Sturegard, E., Johnsson, A., Gustafsson, E. & Dillner, J.(2008). Condyloma typing important for follow up of HPV vaccination. A condyloma reporting project. Lakartidningen 105, 3648–3650 (in Swedish). [Google Scholar]
  44. Walboomers, J. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J. A., Shah, K. V., Snijders, P. J., Peto, J., Meijer, C. J. & Muñoz, N.(1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189, 12–19.[CrossRef] [Google Scholar]
  45. Wang, Z., Hansson, B. G., Forslund, O., Dillner, L., Sapp, M., Schiller, J. T., Bjerre, B. & Dillner, J.(1996). Cervical mucus antibodies against human papillomavirus type 16, 18, and 33 capsids in relation to presence of viral DNA. J Clin Microbiol 34, 3056–3062. [Google Scholar]
  46. Wang, X., Sapp, M., Christensen, N. D. & Dillner, J.(2005). Heparin-based ELISA reduces background reactivity in virus-like particle-based papillomavirus serology. J Gen Virol 86, 65–73.[CrossRef] [Google Scholar]
  47. Waterboer, T., Sehr, P., Michael, K. M., Franceschi, S., Nieland, J. D., Joos, T. O., Templin, M. F. & Pawlita, M.(2005). Multiplex human papillomavirus serology based on in situ-purified glutathione S-transferase fusion proteins. Clin Chem 51, 1845–1853.[CrossRef] [Google Scholar]

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