Expression of a recombinant Human papillomavirus 16 E6GT oncoprotein fused to N- and C-termini of Potato virus X coat protein in Nicotiana benthamiana

  • Noemi Cerovska
  • Tomas Moravec
  • Hana Hoffmeisterova
  • Helena Plchova
  • Helena Synkova
  • Ingrid Polakova
  • Martina Duskova
  • Michal Smahel
Original Paper


Infection with Human papillomaviruses (HPVs) is linked to cervical cancer, which is one of the most common cancers found among women worldwide. Despite preventive immunization, a therapeutic vaccine, targeting infected individuals, is required. Vaccine strategies for treatment of HPV—induced cervical cancer are based on E7 and E6 oncoproteins. In this work we report the transient expression of chimeric particles containing the E6 oncoprotein from Human papillomavirus type 16 (HPV-16) in plants. We fused a mutagenized coding sequence of the E6 oncoprotein (E6GT) with the coding sequence of Potato virus X coat protein (PVX CP) both with the 5′- and 3′-terminus using linkers of different length (0, 4 or 15 amino acids). The expression in E. coli was performed to assess the characteristics of the recombinant protein prior to plant expression. The yield and immunological reactivity of the expressed proteins were screened with anti-PVX CP and E6 antibodies. The highest yields of chimeric particles were observed in the transgenic N. benthamiana expressing Potato virus A HC-Pro protein and the Tobacco mosaic virus movement protein. When inoculated on host plants, these recombinant viruses were not able to spread systemically. The obtained results revealed the new relations for design of expression cassettes for plant-based vaccine production.


Transient expression Molecular farming Potato virus X Human papillomavirus Oncoprotein E6 Bacterial expression 


  1. Alfthan K, Takkinen K, Sizmann D, Soderlund H, Teeri TT (1995) Properties of a single-chain antibody containing different linker peptides. Protein Eng 8:725–731PubMedCrossRefGoogle Scholar
  2. An G (1987) Binary Ti vectors for plant transformation and promoter analysis. Methods Enzymol 153:292–305CrossRefGoogle Scholar
  3. Anwar N, Watanabe KN, Watanabe JA (2011) Transgenic sweet potato expressing mammalian cytochrome P450. Plant Cell Tiss Organ Cult 105:219–231CrossRefGoogle Scholar
  4. Biemelt S, Sonnewald U, Galmbacher P, Willmitzer L, Muller M (2003) Production of human papillomavirus type 16 virus-like particles in transgenic plants. J Virol 77:9211–9220PubMedCrossRefGoogle Scholar
  5. Billich A (2003) HPV vaccine MedImmune/GlaxoSmithKline. Curr Opin Investig Drugs 4:210–213PubMedGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  7. Canizares MC, Nicholson L, Lomonossoff GP (2005) Use of viral vectors for vaccine production in plants. Immunol Cell Biol 83:263–270PubMedCrossRefGoogle Scholar
  8. Cerovska N, Pecenkova T, Moravec T, Veleminsky J (2004) Transient expression of heterologous model gene in plants using Potato virus X-based vector. Plant Cell Tissue Organ Cult 79:147–152CrossRefGoogle Scholar
  9. Cerovska N, Hoffmeisterova H, Pecenkova T, Moravec T, Synkova H, Plchova H, Ludvikova V, Smahel MK (2008) Transient expression of HPV16 E7 peptide (aa 44–60) and HPV16 L2 peptide (aa 108–120) on chimeric potyvirus-like particles using Potato virus X-based vector. Protein Expr Purif 58:154–161PubMedCrossRefGoogle Scholar
  10. Cerovska N, Hoffmeisterova H, Moravec T, Plchova H, Folwarczna J, Synkova H, Ryslava H, Ludvikova V, Smahel M (2012) Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants. J Biosci 37:125–133PubMedCrossRefGoogle Scholar
  11. Chapman S, Kavanagh TA, Baulcombe DC (1992) Potato virus X as a vector for gene expression in plants. Plant J 2:549–557PubMedGoogle Scholar
  12. De la Rosa GP, Monroy-García A, de Lourdes Mora-García M, Reynaga Peńa CG, Hernández-Montes J, Weiss-Steider B, Gómez Lim MA (2009) An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice. Virol J 6:2. doi:10.1186/1743-422X-6-2 CrossRefGoogle Scholar
  13. Eiben GL, Da Silva DM, Steven DM, Fausch C, Le Poole IC, Nishimura MI, Kast WM (2003) Cervical cancer vaccines: recent advances in HPV research. Viral Immunol 16:111–121PubMedCrossRefGoogle Scholar
  14. Faye L, Gomord V (2010) Success stories in molecular farming—a brief overview. Plant Biotechnol J 8:525–528PubMedCrossRefGoogle Scholar
  15. Fedorkin ON, Solovyev AG, Yelina NE, Zamyatin AA, Inovkin RA, Makinen K, Schieman J, Syu M (2001) Cell-to-cell movement of Potato virus X involves distinct functions of the coat protein. J Gen Virol 82:449–458PubMedGoogle Scholar
  16. Fernández-San Millán A, Ortigosa SM, Hervás-Stubbs S, Corral-Martínez P, Seguí-Simarro JM, Gaétan J, Coursaget P, Veramendi J (2008) Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic. Plant Biotechnol J 6:427–441PubMedCrossRefGoogle Scholar
  17. Franconi R, DiBonito P, Dibello F, Accardi L, Muller A, Cirilli A (2002) Plant derived human papillomavirus 16 E7 oncoprotein induces immune response and specific tumor protection. Cancer Res 62:3654–3658PubMedGoogle Scholar
  18. Frazer H (2004) Prevention of cervical cancer through papillomavirus vaccination. Nat Rev Immunol 4:46–54PubMedCrossRefGoogle Scholar
  19. Ganguly N, Parihar SP (2009) Human papillomavirus E6 and E7 oncoproteins as risk factors for tumorigenesis. J Biosci 34:113–123PubMedCrossRefGoogle Scholar
  20. Harper DM, Franco EL, Wheeler CM, Moscicki AB, Romanowski B, Roteli-Martins CM, Jenkins D, Schuind A, Costa Clemens SA, Dubin G (2006) Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial, HPV Vaccine Study group. Lancet 367:1247–1255PubMedCrossRefGoogle Scholar
  21. Hellner K, Munger K (2011) Human papillomaviruses as therapeutic targets in human cancer. J Clin Oncol 29:1785–1794PubMedCrossRefGoogle Scholar
  22. Hirano H, Watanabe T (1990) Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein. Electrophoresis 11:573–580PubMedCrossRefGoogle Scholar
  23. Hoffmeisterova H, Cerovska N, Moravec T, Plchova H, Folwarczna J, Veleminsky J (2008) Transient expression of fusion gene coding for the HPV-16 epitopes fused to the sequence of potyvirus coat protein using different means of inoculation of Nicotiana benthamiana and Brassica rapa, cv. Rapa plants. Plant Cell Tissue Organ Cult 94:261–267CrossRefGoogle Scholar
  24. Hoffmeisterova H, Moravec T, Plchova H, Folwarczna J, Cerovska N (2012) The influence of the N- and C-terminal modifications of Potato virus X coat protein on virus properties. Biol Plant 56:775–779CrossRefGoogle Scholar
  25. Hsu CH, Peng KL, Jhang HC, Lin CH, Wu SY, Chiang CM, Lee SC, Yu WC, Juan LJ (2011) The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription. Oncogene 31:2335–2349PubMedCrossRefGoogle Scholar
  26. Kim MY, Kim TG, Yoo HS, Yang MS (2011) Expression and assembly of ApxIIA toxin of Actinobacillus pleuropneumoniae fused with the enterotoxigenic E. coli heat-labile toxin B subunit in transgenic tobacco. Plant Cell Tissue Organ Cult 105:375–382CrossRefGoogle Scholar
  27. Koutsky LA, Ault KA, Wheeler CM, Brown DR, Bar E, Alvarez FB, Chiacchierini LM, Jansen KU (2002) A controlled trial of a human papillomavirus type 16 vaccine. Proof of principle study investigators. N Engl J Med 347:1645–1651PubMedCrossRefGoogle Scholar
  28. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  29. Lico C, Capuano F, Renzone G, Donini M, Marusic C, Scaloni A, Benvenuto E, Baschieri S (2006) Peptide display on Potato virus X: molecular features of the coat protein- fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles. J Gen Virol 87:3103–3112PubMedCrossRefGoogle Scholar
  30. Lico C, Mancinia C, Italiani P, Betti C, Boraschib D, Benvenuto E, Baschieri S (2009) Plant-produced Potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice. Vaccine 27:5069–5076PubMedCrossRefGoogle Scholar
  31. Liu HL, Li WS, Lei T, Zheng J, Zhang Z, Yan XF, Wang ZZ, Wang IL, Si LS (2005) Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants. Acta Biochim Biophys Sin 37:153–158PubMedGoogle Scholar
  32. Ma B, Xu Y, Hung C-F, Wu T-C (2010) HPV and therapeutic vaccines: where are we in 2010? Curr Cancer Ther Rev 6:81–103CrossRefGoogle Scholar
  33. Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, Fischer R, Williamson AL, 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
  34. Marconi G, Albertini E, Barone P, De Marchis F, Lico C, Marusic C, Rutili D, Veronesi F, Porceddu A (2009) In planta production of two peptides of the Classical Swine Fever Virus (CSFV) E2 glycoprotein fused to the coat protein of Potato virus X. BMC Biotechnol 6:1–9Google Scholar
  35. Martínez-González L, Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, López-Revilla R, Korban SS, Guevara-Arauza JC, Alpuche-Solís AG (2011) Oral immunization with a lettuce-derived Escherichia coli heat-labile toxin B subunit induces neutralizing antibodies in mice. Plant Cell Tiss Organ Cult 107:441–449CrossRefGoogle Scholar
  36. Marusic C, Rizza P, Lattanzi L, Mancini C, Spada M, Belardelli F, Benvenuto E, Capone I (2001) Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J Virol 75:8434–8439PubMedCrossRefGoogle Scholar
  37. Massa S, Simeone P, Muller A, Benvenuto E, Venuti A, Franconi R (2008) Antitumor activity of DNA vaccines based on the human papillomavirus-16 E7 protein genetically fused to a plant virus coat protein. Hum Gene Ther 19:354–364PubMedCrossRefGoogle Scholar
  38. McLaughlin-Drubin ME, Munger K (2009) Oncogenic activities of human papillomaviruses. Virus Res 143:195–208PubMedCrossRefGoogle Scholar
  39. Morgenfeld M, Segretin ME, Wirth S, Lentz E, Zelada A, Mentaberry A, Glossmann L, Almonacid FB (2009) Potato virus X coat protein fusion to human papillomavirus 16 E7 oncoprotein enhance antigen stability and accumulation in tobacco chloroplast. Mol Biotechnol 43:243–249PubMedCrossRefGoogle Scholar
  40. Mowat WP (1985) Simplified enzyme immunoassay for plant virus detection and identification. In: Harrison BD, Murant AF (eds) Report of the Scottish crop Researche Institute for 1984. Scottish Crop Research Institute, Dundee, p 188Google Scholar
  41. Münger K, Phelps WC, Bubb V, Howley PM, Schlegel R (1989) The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primer human keratinocytes. J Virol 63:4417–4421PubMedGoogle Scholar
  42. Plchova H, Moravec T, Hoffmeisterova H, Folwarczna J, Cerovska N (2011) Expression of Human papillomavirus 16 E7ggg oncoprotein on N- and C-terminus of Potato virus X coat protein in bacterial and plant cells. Protein Expr Purif 77:146–152PubMedCrossRefGoogle Scholar
  43. Pokorná D, Čeřovská N, Šmahel M, Moravec T, Ludvíková V, Macková J, Synková H, Dušková M, Hozák P, Velemínský J (2005) DNA vaccines based on chimeric potyvirus-like particles carrying HPV16 E7 peptide (aa 44–60). Oncol Rep 14:1045–1053PubMedGoogle Scholar
  44. Polakova I, Pokorna D, Duskova M, Smahel M (2010) DNA vaccine against human papillomavirus type 16: modifications of the E6 oncogene. Vaccine 28:1506–1513PubMedCrossRefGoogle Scholar
  45. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  46. Scholthof K-BG, Mirkov TE, Scholthof HB (2002) Plant viral gene vectors: biotechnology applications in agriculture and medicine. Genet Eng 24:67–85CrossRefGoogle Scholar
  47. Tindle RW (2002) Immune evasion in human papillomavirus-associated cervical cancer. Nature Rev Cancer 2:59–65CrossRefGoogle Scholar
  48. Uhde-Holzem K, Fischer R, Commandeur U (2007) Genetic stability of recombinant potato virus X virus vectors presenting foreign epitopes. Arch Virol 152:805–811PubMedCrossRefGoogle Scholar
  49. Varsani A, Williamson AL, Rose RC, Jaffer M, Rybicki EP (2003) Expression of Human papillomavirus type 16 major capsid protein in transgenic Nicotiana tabacum cv, Xanthi. Arch Virol 148:1771–1786PubMedCrossRefGoogle Scholar
  50. Villa LL, Costa RL, Petta CA, Andrade RP, Ault KA, Giuliano AR, Wheeler CM, Koutsky LA, Malm C, Lehtinen M, Skjeldestad FE, Olsson SE, Steinwall M, Brown DR, Kurman RJ, Ronnett BM, Stoler MH, Ferenczy A, Harper DM, Tamms GM, Yu J, Lupinacci L, Railkar R, Taddeo FJ, Jansen KU, Esser MT, Sings HL, Saah AJ, Barr E (2005) Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial. Lancet Oncol 6:271–278PubMedCrossRefGoogle Scholar
  51. Warens AN, Jones MD, Lechler RI (1997) Splicing by overlap extension by PCR using asymmetric amplification improved technique for the generation of hybrid proteins of immunological interest. Gene 186:29–35CrossRefGoogle Scholar
  52. Woodland DL (2004) Jump-starting the immune system: prime-boosting comes of age. Trends Immunol 25:98–103PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Noemi Cerovska
    • 1
  • Tomas Moravec
    • 1
  • Hana Hoffmeisterova
    • 1
  • Helena Plchova
    • 1
  • Helena Synkova
    • 1
  • Ingrid Polakova
    • 2
  • Martina Duskova
    • 2
  • Michal Smahel
    • 2
  1. 1.Institute of Experimental Botany, v. v. i.Academy of Sciences of the Czech RepublicPrague 6Czech Republic
  2. 2.Department of Experimental VirologyInstitute of Hematology and Blood TransfusionPrague 2Czech Republic

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