title: Preclinical safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7.
abstract: Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of 'gene-shuffled' (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.
authors.affiliation: Department of Pathology, Unit of Molecular Pathology, VU University Medical Center, PO Box 7057, 1007 MB, Amsterdam, The Netherlands.
authors.forename: F E
authors.initials: FE
authors.lastname: Henken
authors.forename: K
authors.initials: K
authors.lastname: Oosterhuis
authors.forename: P
authors.initials: P
authors.lastname: Öhlschläger
authors.forename: L
authors.initials: L
authors.lastname: Bosch
authors.forename: E
authors.initials: E
authors.lastname: Hooijberg
authors.forename: J B A G
authors.initials: JB
authors.lastname: Haanen
authors.forename: R D M
authors.initials: RD
authors.lastname: Steenbergen
mesh_terms: D000818:Animals; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D032621:DNA Shuffling; D015870:Gene Expression; D006801:Humans; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins / Q000235:genetics / Q000378:metabolism*; D053918:Papillomavirus Vaccines / Q000008:administration & dosage / Q000009:adverse effects* / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013745:Tetanus Toxoid / Q000009:adverse effects / Q000235:genetics; D014161:Transduction, Genetic; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000009:adverse effects* / Q000235:genetics; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D053918:Papillomavirus Vaccines; D012097:Repressor Proteins; D013745:Tetanus Toxoid; D019444:Vaccines, DNA; D014614:Vaccines, Synthetic; C059731:oncogene protein E7, Human papillomavirus type 16