title: Codon optimized expression of HPV 16 E6 renders target cells susceptible to E6-specific CTL recognition.
abstract: The early proteins E6 and E7 of the cancer-related human papillomavirus type 16 (HPV 16) are constitutively expressed in cancer cells thus are targets for immune therapeutic approaches. Whereas previous studies have mainly focussed on the immunogenicity of E7 protein little is known about E6. In order to evaluate E6-specific DNA immunization strategies in a preclinical mouse model C57BL/6 mice were injected with plasmid pTHampE6 and analyzed for E6-specific CTL induction. CTL specific for the H2-K(b)-restricted E6-derived epitope E6 48-57, were readily detectable among splenocytes of immunized animals, however, these CTL showed a differential recognition pattern on various E6-expressing target cells. Using a newly generated E6-specific monoclonal antibody we found that most cell lines expressing E6 encoded by the natural gene showed undetectable protein amounts and were ignored by E6-specific CTL. However, transfection of a codon optimized version of the E6 gene (E6opt) strongly enhanced protein expression levels within these cells turning them into susceptible target cells. Surprisingly, we found that E6-positive TC-1 cells, although recognized by E6-specific CTL, were totally devoid of any detectable E6 protein. Inhibition of proteasomal function by lactacystin treatment diminished E6-specific CTL recognition of TC-1 cells and RMA/E6opt transfectants accompanied by intracellular accumulation of E6 protein as observed in RMA/E6opt transfectants, but not in TC-1 cells. These data suggest that in TC-1 cells rapid degradation processes might prevent stable expression of E6 protein yet generate precursor peptides in amounts sufficient for MHC class I restricted antigen presentation. Thus, the results presented in this paper show that: (i) use of optimized codons in transfection experiments can improve susceptibility of target cells to E6-specific CTL recognition and (ii) lack of detectable protein within a cell does not necessarily indicate the absence of epitope presentation. Both findings are of potential relevance for the design of tumor vaccines.
authors.affiliation: Deutsches Krebsforschungszentrum, Heidelberg, Forschungsschwerpunkt Infektionen und Krebs, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. r.samorski@dkfz.de
authors.forename: Regina
authors.initials: R
authors.lastname: Samorski
authors.forename: Lutz
authors.initials: L
authors.lastname: Gissmann
authors.forename: Wolfram
authors.initials: W
authors.lastname: Osen
mesh_terms: D000818:Animals; D019496:Cancer Vaccines / Q000276:immunology; D003062:Codon; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005260:Female; D005813:Genes, Synthetic / Q000276:immunology; D052162:Human papillomavirus 16 / Q000276:immunology; D016233:Immunotherapy, Active / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology*; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000821:virology*; D014162:Transfection / Q000379:methods; D019444:Vaccines, DNA / Q000276:immunology*; D014765:Viral Vaccines / Q000276:immunology*
chemical_list: D019496:Cancer Vaccines; D003062:Codon; C068651:E6 protein, Human papillomavirus type 16; D018984:Epitopes, T-Lymphocyte; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D019444:Vaccines, DNA; D014765:Viral Vaccines