title: Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli beta-glucuronidase.
abstract: BACKGROUND
  Human papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity. In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb-binding site of the HPV16 E7 protein.
  
  
  RESULTS
  To increase E7GGG immunogenicity we constructed fusion genes of E. coli beta-glucuronidase (GUS) with one or three copies of E7GGG. Furthermore, a similar construct was prepared with partial E7GGG (E7GGGp, 41 amino acids from the N-terminus). The expression of the fusion genes was examined in human 293T cells. Quantification of GUS activity and the amount of E7 antigen showed substantially reduced GUS activity of fusion proteins with complete E7GGG that was mainly caused by decrease of their steady-state level in comparison with GUS or E7GGGpGUS. Still, the steady-state level of E7GGG.GUS was about 20-fold higher than that of the E7GGG protein. The immunogenicity of the fusion genes with complete E7GGG was tested by DNA immunisation of C57BL/6 mice with a gene gun. TC-1 cells and their clone TC-1/A9 with down-regulated MHC class I expression were subcutaneously (s.c.) inoculated to induce tumour formation. All mice were protected against challenge with TC-1 cells and most animals remained tumour-free in therapeutic-immunisation experiments with these cells, in contrast to immunisation with unfused E7GGG and the fusion with the lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Significant protection was also recorded against TC-1/A9 cells. Both tetramer staining and ELISPOT assay showed substantially higher activation of E7-specific CD8+ lymphocytes in comparison with E7GGG and Sig/E7GGG/LAMP-1. Deletion of 231 bp in the GUS gene eliminated enzymatic activity, but did not influence the immunogenicity of the E7GGG.GUS gene.
  
  
  CONCLUSIONS
  The findings demonstrate the superior immunisation efficacy of the fusion genes of E7GGG with GUS when compared with E7GGG and Sig/E7GGG/LAMP-1. The E7GGG.GUS-based DNA vaccine might also be efficient against human tumour cells with reduced MHC class I expression.
authors.affiliation: Institute of Hematology and Blood Transfusion, Department of Experimental Virology, Prague, Czech Republic. smahel@uhkt.cz
authors.forename: Michal
authors.initials: M
authors.lastname: Smahel
authors.forename: Dana
authors.initials: D
authors.lastname: Pokorná
authors.forename: Jana
authors.initials: J
authors.lastname: Macková
authors.forename: Josef
authors.initials: J
authors.lastname: Vlasák
mesh_terms: D000818:Animals; D001665:Binding Sites; D019470:Biolistics; D015152:Blotting, Northern; D018414:CD8-Positive T-Lymphocytes; D002460:Cell Line; D045744:Cell Line, Tumor; D002472:Cell Transformation, Viral; D004926:Escherichia coli / Q000201:enzymology*; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D012313:RNA / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D019444:Vaccines, DNA
chemical_list: D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D010455:Peptides; D019444:Vaccines, DNA; C059731:oncogene protein E7, Human papillomavirus type 16; D012313:RNA; D005966:Glucuronidase