Archives of Virology

, Volume 158, Issue 10, pp 2079–2088 | Cite as

Development of human papillomavirus chimaeric L1/L2 candidate vaccines

  • Marieta McGrath
  • Gillian K. de Villiers
  • Enid Shephard
  • Inga I. Hitzeroth
  • Edward P. Rybicki
Original Article


Recombinant human papillomavirus (HPV) virus-like particle (VLP) vaccines based on the L1 capsid protein have been shown to be efficient prophylactic vaccines, albeit type-specific. As a first step to investigate the feasibility of extending protection against non-vaccine types, HPV-16 L1 chimaeras were generated. The region downstream of L1 amino acid (aa) 413 was replaced with selected cross-neutralising epitopes (aa 108-120; 56-81 and 17-36) derived from the HPV-16 L2 protein, generating proteins designated SAF, L2.56 and L2.17, respectively. The chimaera L1BPV containing BPV-1 L2 peptide aa 1-88 was similarly constructed. The chimaeras were evaluated for expression in insect cells; their ability to form particles was studied by electron microscopy, and their immunogenicity was evaluated in mice. SAF, L2.56 and L2.17 proteins were expressed to high concentrations in insect cells and elicited HPV-16 pseudovirus-neutralising anti-L1 antibodies. L2.56 and L2.17 also elicited anti-L2 antibodies. L1BPV was a poor vaccine candidate due to low levels of expression with concomitant lack of immunogenicity. All chimaeras assembled into tertiary structures. The results indicate that chimaeric L1 vaccines incorporating cross-neutralising L2 peptides could be promising second-generation prophylactic HPV vaccine candidates.


  1. 1.
    Alphs HH, Gambhira R, Karanam B, Roberts JN, Jagu S, Schiller JT, Zeng W, Jackson DC, Roden RBS (2008) Protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of L2. Proc Natl Acad Sci USA 105:5850–5855PubMedCrossRefGoogle Scholar
  2. 2.
    Bernard H-U, Burk RD, Chen Z, Van Doorslaer K, Hausen Hz, De Villiers E-M (2010) Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology 401:70–79PubMedCrossRefGoogle Scholar
  3. 3.
    Bishop B, Dasgupta J, Chen XS (2007) Structure-based engineering of papillomavirus major capsid L1: controlling particle assembly. Virol J 4:3PubMedCrossRefGoogle Scholar
  4. 4.
    Bosch FX (2009) Broad-spectrum human papillomavirus vaccines: new horizons but one step at a time. J Natl Cancer Inst 101:771–773PubMedCrossRefGoogle Scholar
  5. 5.
    Bosch FX, Burchell AN, Schiffman M, Giuliano AR, De Sanjose S, Bruni L, Tortolero-Luna G, Kjaer SK, Muñoz N (2008) Epidemiology and natural history of human papillomavirus infections and type-specific implications in cervical neoplasia. Vaccine 26(Suppl 10):K1–K16PubMedCrossRefGoogle Scholar
  6. 6.
    Breitburd F, Kirnbauer R, Hubbert NL, Nonnenmacher B, Trin-Dinh-Desmarquet C, Orth G, Schiller JT, Lowy DR (1995) Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection. J Virol 69:3959–3963PubMedGoogle Scholar
  7. 7.
    Buck CB, Cheng N, Thompson CD, Lowy DR, Steven AC, Schiller JT, Trus BL (2008) Arrangement of L2 within the papillomavirus capsid. J Virol 82:5190–5197PubMedCrossRefGoogle Scholar
  8. 8.
    Buck CB, Pastrana DV, Lowy DR, Schiller JT (2005) Generation of HPV pseudovirions using transfection and their use in neutralization assays. Methods Mol Med 119:445–462PubMedGoogle Scholar
  9. 9.
    Cason J, Kambo PK, Jewers RJ, Best JM (1994) Detection of protein aggregates, but not virus-like particles, when the major (L1) coat protein of a wild-type human papillomavirus type 16 (HPV-16) is expressed in insect cells. Biochem Soc Trans 22:335SPubMedGoogle Scholar
  10. 10.
    Castellsagué X (2008) Natural history and epidemiology of HPV infection and cervical cancer. Gynecol Oncol 110:S4–S7PubMedCrossRefGoogle Scholar
  11. 11.
    Chackerian B, Lowy DR, Schiller JT (1999) Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles. Proc Natl Acad Sci USA 96:2373–2378PubMedCrossRefGoogle Scholar
  12. 12.
    Chen XS, Garcea RL, Goldberg I, Casini G, Harrison SC (2000) Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16. Mol Cell 5:557–567PubMedCrossRefGoogle Scholar
  13. 13.
    Christensen ND, Dillner J, Eklund C, Carter JJ, Wipf GC, Reed CA, Cladel NM, Galloway DA (1996) Surface conformational and linear epitopes on HPV-16 and HPV-18 L1 virus-like particles as defined by monoclonal antibodies. Virology 223:174–184PubMedCrossRefGoogle Scholar
  14. 14.
    Christensen ND, Kreider JW (1990) Antibody-mediated neutralization in vivo of infectious papillomaviruses. J Virol 64:3151–3156PubMedGoogle Scholar
  15. 15.
    Cutts FT, Franceschi S, Goldie S, Castellsague X, De Sanjose S, Garnett G, Edmunds WJ, Claeys P, Goldenthal KL, Harper DM, Markowitz L (2007) Human papillomavirus and HPV vaccines: a review. Bull World Health Organ 85:719–726PubMedCrossRefGoogle Scholar
  16. 16.
    Deschuyteneer M, Elouahabi A, Plainchamp D, Plisnier M, Soete D, Corazza Y, Lockman L, Giannini S, Deschamps M (2010) Molecular and structural characterization of the L1 virus-like particles that are used as vaccine antigens in CervarixTM, the AS04-adjuvanted HPV-16 and -18 cervical cancer vaccine. Hum Vaccin 6:407–419PubMedCrossRefGoogle Scholar
  17. 17.
    de Villiers E-M, Fauquet C, Broker TR, Bernard H-U, Zur Hausen H (2004) Classification of papillomaviruses. Virology 324:17–27PubMedCrossRefGoogle Scholar
  18. 18.
    Embers ME, Budgeon LR, Pickel M, Christensen ND (2002) Protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of L2, the minor capsid protein. J Virol 76:9798–9805PubMedCrossRefGoogle Scholar
  19. 19.
    Fang NX, Frazer IH, Fernando GJ (2000) Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system. Biotechnol Appl Biochem 32(Pt 1):27–33PubMedCrossRefGoogle Scholar
  20. 20.
    Gambhira R, Karanam B, Jagu S, Roberts JN, Buck CB, Bossis I, Alphs H, Culp T, Christensen ND, Roden RBS (2007) A protective and broadly cross-neutralizing epitope of human papillomavirus L2. J Virol 81:13927–13931PubMedCrossRefGoogle Scholar
  21. 21.
    Huh WK, Roden RBS (2008) The future of vaccines for cervical cancer. Gynecol Oncol 109:S48–S56PubMedCrossRefGoogle Scholar
  22. 22.
    Kawana K, Matsumoto K, Yoshikawa H, Taketani Y, Kawana T, Yoshiike K, Kanda T (1998) A surface immunodeterminant of human papillomavirus type 16 minor capsid protein L2. Virology 245:353–359PubMedCrossRefGoogle Scholar
  23. 23.
    Kawana K, Yasugi T, Kanda T, Kino N, Oda K, Okada S, Kawana Y, Nei T, Takada T, Toyoshima S, Tsuchiya A, Kondo K, Yoshikawa H, Tsutsumi O, Taketani Y (2003) Safety and immunogenicity of a peptide containing the cross-neutralization epitope of HPV16 L2 administered nasally in healthy volunteers. Vaccine 21:4256–4260PubMedCrossRefGoogle Scholar
  24. 24.
    Kawana K, Yoshikawa H, Taketani Y, Yoshiike K, Kanda T (1999) Common neutralization epitope in minor capsid protein L2 of human papillomavirus types 16 and 6. J Virol 73:6188–6190PubMedGoogle Scholar
  25. 25.
    Kirnbauer R, Booy F, Cheng N, Lowy DR, Schiller JT (1992) Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci USA 89:12180–12184PubMedCrossRefGoogle Scholar
  26. 26.
    Koh YT, Higgins SA, Weber JS, Kast WM (2006) Immunological consequences of using three different clinical/laboratory techniques of emulsifying peptide-based vaccines in incomplete Freund’s adjuvant. J Transl Med 4:42PubMedCrossRefGoogle Scholar
  27. 27.
    Kohl TO, Hitzeroth II, Christensen ND, Rybicki EP (2007) Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum. BMC Biotechnol 7:56PubMedCrossRefGoogle Scholar
  28. 28.
    Kondo K, Ishii Y, Ochi H, Matsumoto T, Yoshikawa H, Kanda T (2007) Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region. Virology 358:266–272PubMedCrossRefGoogle Scholar
  29. 29.
    Kondo K, Ochi H, Matsumoto T, Yoshikawa H, Kanda T (2008) Modification of human papillomavirus-like particle vaccine by insertion of the cross-reactive L2-epitopes. J Med Virol 80:841–846PubMedCrossRefGoogle Scholar
  30. 30.
    Kuck D, Leder C, Kern A, Müller M, Piuko K, Gissmann L, Kleinschmidt JA (2006) Efficiency of HPV 16 L1/E7 DNA immunization: influence of cellular localization and capsid assembly. Vaccine 24:2952–2965PubMedCrossRefGoogle Scholar
  31. 31.
    Lenz P, Day PM, Pang YY, Frye SA, Jensen PN, Lowy DR, Schiller JT (2001) Papillomavirus-like particles induce acute activation of dendritic cells. J Immunol 166:5346–5355PubMedGoogle Scholar
  32. 32.
    Lenz P, Thompson CD, Day PM, Bacot SM, Lowy DR, Schiller JT (2003) Interaction of papillomavirus virus-like particles with human myeloid antigen-presenting cells. Clin Immunol 106:231–237PubMedCrossRefGoogle Scholar
  33. 33.
    Liu XS, Liu WJ, Zhao KN, Liu YH, Leggatt G, Frazer IH (2002) Route of administration of chimeric BPV1 VLP determines the character of the induced immune responses. Immunol Cell Biol 80:21–29PubMedCrossRefGoogle Scholar
  34. 34.
    Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, Fischer R, Williamson A-L, Rybicki EP (2007) Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization. J Gen Virol 88:1460–1469PubMedCrossRefGoogle Scholar
  35. 35.
    Murata Y, Lightfoote PM, Rose RC, Walsh EE (2009) Antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix 4 loop region of human papillomavirus L1 capsomeres. Virol J 6:81PubMedCrossRefGoogle Scholar
  36. 36.
    Neeper MP, Hofmann KJ, Jansen KU (1996) Expression of the major capsid protein of human papillomavirus type 11 in Saccharomyces cerevisae. Gene 180:1–6PubMedCrossRefGoogle Scholar
  37. 37.
    Ohlschläger P, Osen W, Dell K, Faath S, Garcea RL, Jochmus I, Müller M, Pawlita M, Schäfer K, Sehr P, Staib C, Sutter G, Gissmann L (2003) Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice. J Virol 77:4635–4645PubMedCrossRefGoogle Scholar
  38. 38.
    Pastrana DV, Buck CB, Pang Y-YS, Thompson CD, Castle PE, FitzGerald PC, Krüger Kjaer S, Lowy DR, Schiller JT (2004) Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18. Virology 321:205–216PubMedCrossRefGoogle Scholar
  39. 39.
    Pastrana DV, Gambhira R, Buck CB, Pang Y-YS, Thompson CD, Culp TD, Christensen ND, Lowy DR, Schiller JT, Roden RBS (2005) Cross-neutralization of cutaneous and mucosal Papillomavirus types with anti-sera to the amino terminus of L2. Virology 337:365–372PubMedCrossRefGoogle Scholar
  40. 40.
    Pillay S, Meyers A, Williamson A-L, Rybicki EP (2009) Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression system. Biotechnol Prog 25:1153–1160PubMedCrossRefGoogle Scholar
  41. 41.
    Roden RB, Yutzy WH 4th, Fallon R, Inglis S, Lowy DR, Schiller JT (2000) Minor capsid protein of human genital papillomaviruses contains subdominant, cross-neutralizing epitopes. Virology 270:254–257PubMedCrossRefGoogle Scholar
  42. 42.
    Rose RC, Bonnez W, Da Rin C, McCance DJ, Reichman RC (1994) Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles. J Gen Virol 75(Pt 9):2445–2449PubMedCrossRefGoogle Scholar
  43. 43.
    Rubio I, Bolchi A, Moretto N, Canali E, Gissmann L, Tommasino M, Müller M, Ottonello S (2009) Potent anti-HPV immune responses induced by tandem repeats of the HPV16 L2 (20–38) peptide displayed on bacterial thioredoxin. Vaccine 27:1949–1956PubMedCrossRefGoogle Scholar
  44. 44.
    Rudolf MP, Fausch SC, Da Silva DM, Kast WM (2001) Human dendritic cells are activated by chimeric human papillomavirus type-16 virus-like particles and induce epitope-specific human T cell responses in vitro. J Immunol 166:5917–5924PubMedGoogle Scholar
  45. 45.
    Schädlich L, Senger T, Gerlach B, Mücke N, Klein C, Bravo IG, Müller M, Gissmann L (2009) Analysis of modified human papillomavirus type 16 L1 capsomeres: the ability to assemble into larger particles correlates with higher immunogenicity. J Virol 83:7690–7705PubMedCrossRefGoogle Scholar
  46. 46.
    Schellenbacher C, Roden R, Kirnbauer R (2009) Chimeric L1–L2 virus-like particles as potential broad-spectrum human papillomavirus vaccines. J Virol 83:10085–10095PubMedCrossRefGoogle Scholar
  47. 47.
    Schiller JT, Castellsagué X, Villa LL, Hildesheim A (2008) An update of prophylactic human papillomavirus L1 virus-like particle vaccine clinical trial results. Vaccine 26(Suppl 10):K53–K61PubMedCrossRefGoogle Scholar
  48. 48.
    Senger T, Schädlich L, Gissmann L, Müller M (2009) Enhanced papillomavirus-like particle production in insect cells. Virology 388:344–353PubMedCrossRefGoogle Scholar
  49. 49.
    Shi L, Sings HL, Bryan JT, Wang B, Wang Y, Mach H, Kosinski M, Washabaugh MW, Sitrin R, Barr E (2007) GARDASIL: prophylactic human papillomavirus vaccine development–from bench top to bed-side. Clin Pharmacol Ther 81:259–264PubMedCrossRefGoogle Scholar
  50. 50.
    Slupetzky K, Shafti-Keramat S, Lenz P, Brandt S, Grassauer A, Sara M, Kirnbauer R (2001) Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops. J Gen Virol 82:2799–2804PubMedGoogle Scholar
  51. 51.
    Thönes N, Herreiner A, Schädlich L, Piuko K, Müller M (2008) A direct comparison of human papillomavirus type 16 L1 particles reveals a lower immunogenicity of capsomeres than viruslike particles with respect to the induced antibody response. J Virol 82:5472–5485PubMedCrossRefGoogle Scholar
  52. 52.
    Touze A, El Mehdaoui S, Sizaret PY, Mougin C, Muñoz N, Coursaget P (1998) The L1 major capsid protein of human papillomavirus type 16 variants affects yield of virus-like particles produced in an insect cell expression system. J Clin Microbiol 36:2046–2051PubMedGoogle Scholar
  53. 53.
    Varsani A, Williamson A-L, De Villiers D, Becker I, Christensen ND, Rybicki EP (2003) 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 77:8386–8393PubMedCrossRefGoogle Scholar
  54. 54.
    Wakabayashi MT, Da Silva DM, Potkul RK, Kast WM (2002) Comparison of human papillomavirus type 16 L1 chimeric virus-like particles versus L1/L2 chimeric virus-like particles in tumor prevention. Intervirology 45:300–307PubMedCrossRefGoogle Scholar
  55. 55.
    World Health Organization—International Agency for Research on Cancer (2007) Monograph on Human Papillomaviruses. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol 90Google Scholar
  56. 56.
    Yuan H, Estes PA, Chen Y, Newsome J, Olcese VA, Garcea RL, Schlegel R (2001) Immunization with a pentameric L1 fusion protein protects against papillomavirus infection. J Virol 75:7848–7853PubMedCrossRefGoogle Scholar
  57. 57.
    WHO | Immunization. Accessed 6 Sep 2011

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Marieta McGrath
    • 1
  • Gillian K. de Villiers
    • 1
  • Enid Shephard
    • 2
    • 3
    • 4
  • Inga I. Hitzeroth
    • 1
  • Edward P. Rybicki
    • 2
    • 1
  1. 1.Department of Molecular and Cell BiologyUniversity of Cape TownCape TownSouth Africa
  2. 2.Institute of Infectious Diseases and Molecular MedicineCape TownSouth Africa
  3. 3.Department of Medicine Faculty of Health SciencesUniversity of Cape TownObservatorySouth Africa
  4. 4.Medical Research CouncilTygerbergSouth Africa

Personalised recommendations