title: A prime/boost strategy using DNA/fowlpox recombinants expressing the genetically attenuated E6 protein as a putative vaccine against HPV-16-associated cancers.
abstract: BACKGROUND
  Considering the high number of new cases of cervical cancer each year that are caused by human papilloma viruses (HPVs), the development of an effective vaccine for prevention and therapy of HPV-associated cancers, and in particular against the high-risk HPV-16 genotype, remains a priority. Vaccines expressing the E6 and E7 proteins that are detectable in all HPV-positive pre-cancerous and cancer cells might support the treatment of HPV-related lesions and clear already established tumors.
  
  
  METHODS
  In this study, DNA and fowlpox virus recombinants expressing the E6F47R mutant of the HPV-16 E6 oncoprotein were generated, and their correct expression verified by RT-PCR, Western blotting and immunofluorescence. Immunization protocols were tested in a preventive or therapeutic pre-clinical mouse model of HPV-16 tumorigenicity using heterologous (DNA/FP) or homologous (DNA/DNA and FP/FP) prime/boost regimens. The immune responses and therapeutic efficacy were evaluated by ELISA, ELISPOT assays, and challenge with TC-1* cells.
  
  
  RESULTS
  In the preventive protocol, while an anti-E6-specific humoral response was just detectable, a specific CD8(+) cytotoxic T-cell response was elicited in immunized mice. After the challenge, there was a delay in cancer appearance and a significant reduction of tumor volume in the two groups of E6-immunized mice, thus confirming the pivotal role of the CD8(+) T-cell response in the control of tumor growth in the absence of E6-specific antibodies. In the therapeutic protocol, in-vivo experiments resulted in a higher number of tumor-free mice after the homologous DNA/DNA or heterologous DNA/FP immunization.
  
  
  CONCLUSIONS
  These data establish a preliminary indication for the prevention and treatment of HPV-related tumors by the use of DNA and avipox constructs as safe and effective immunogens following a prime/boost strategy. The combined use of recombinants expressing both E6 and E7 proteins might improve the antitumor efficacy, and should represent an important approach to control HPV-associated cancers.
authors.affiliation: Department of Pharmacological and Biomolecular Sciences, Università di Milano, Milan, Italy. massimiliano.bissa@unimi.it.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Bissa
authors.affiliation: Department of Pharmacological and Biomolecular Sciences, Università di Milano, Milan, Italy. elena.illiano@unimi.it.
authors.forename: Elena
authors.initials: E
authors.lastname: Illiano
authors.affiliation: Department of Medical Biotechnologies and Translational Medicine, Università di Milano, Milan, Italy. sole.pacchioni@studenti.unimi.it.
authors.forename: Sole
authors.initials: S
authors.lastname: Pacchioni
authors.affiliation: Laboratory of Virology HPV-UNIT, Regina Elena National Cancer Institute, Rome, Italy. paolini@ifo.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Paolini
authors.affiliation: Department of Medical Biotechnologies and Translational Medicine, Università di Milano, Milan, Italy. carlo.zanotto@unimi.it.
authors.forename: Carlo
authors.initials: C
authors.lastname: Zanotto
authors.affiliation: Department of Medical Biotechnologies and Translational Medicine, Università di Milano, Milan, Italy. carlo.degiulimorghen@unimi.it.|Cellular and Molecular Pharmacology Section, CNR Institute of Neurosciences, Università di Milano, Milan, Italy. carlo.degiulimorghen@unimi.it.
authors.forename: Carlo
authors.initials: C
authors.lastname: De Giuli Morghen
authors.affiliation: Technical Unit of Radiation Biology and Human Health, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Casaccia Research Centre, Rome, Italy. silvia.massa@enea.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Massa
authors.affiliation: Technical Unit of Radiation Biology and Human Health, Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Casaccia Research Centre, Rome, Italy. rosella.franconi@enea.it.
authors.forename: Rosella
authors.initials: R
authors.lastname: Franconi
authors.affiliation: Department of Pharmacological and Biomolecular Sciences, Università di Milano, Milan, Italy. antonia.radaelli@unimi.it.|Cellular and Molecular Pharmacology Section, CNR Institute of Neurosciences, Università di Milano, Milan, Italy. antonia.radaelli@unimi.it.
authors.forename: Antonia
authors.initials: A
authors.lastname: Radaelli
authors.affiliation: Laboratory of Virology HPV-UNIT, Regina Elena National Cancer Institute, Rome, Italy. venuti@ifo.it.
authors.forename: Aldo
authors.initials: A
authors.lastname: Venuti
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D019496:Cancer Vaccines / Q000276:immunology*; D002642:Chick Embryo; D004274:DNA, Recombinant / Q000378:metabolism*; D005260:Female; D005586:Fowlpox / Q000378:metabolism*; D052162:Human papillomavirus 16 / Q000276:immunology*; D006801:Humans; D056724:Immunity, Humoral / Q000276:immunology; D007117:Immunization, Secondary; D008810:Mice, Inbred C57BL; D009369:Neoplasms / Q000276:immunology* / Q000473:pathology; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D019076:Transgenes; D014611:Vaccination; D014779:Virus Replication
chemical_list: D019496:Cancer Vaccines; D004274:DNA, Recombinant; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins