title: Combined immunization with fusion genes of mutated E7 gene of human papillomavirus type 16 did not enhance antitumor effect.
abstract: BACKGROUND
  The E7 oncoprotein of human papillomavirus type 16 (HPV16) is frequently used as a model tumor-associated antigen. Its immunogenicity has been substantially enhanced by fusion with several proteins of various origins and functions. Different mechanisms have been responsible for increased vaccination efficacy of fusion proteins.
  
  
  RESULTS
  We linked E7 and its mutated form (E7GGG) with the mouse heat-shock protein 70.1 (HSP70.1). Enhanced immunogenicity of both fusion genes administered via a gene gun was demonstrated by protection of C57BL/6 mice against oncogenic MHC class I positive TC-1 cells producing the HPV16 E7 oncoprotein but not against the MHC class I negative TC-1/A9 subline. To assess if the efficacy of E7-based DNA vaccines could be increased by combination of various fusion genes, we combined the HSP70.1 fusion genes (i.e. E7HSP or E7GGGHSP) with the fusion construct linking E7GGG with targeting signals of lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Treatment of mice 4 days after TC-1 cell inoculation showed moderately higher immunization potency of HSP70.1 fusion genes in comparison with the Sig/E7GGG/LAMP-1 gene. Any combination of two fusion genes given in the same gene gun shot neither was more effective compared with single genes nor protected mice against TC-1/A9 cells. As fusion of E7GGG with E. coli glucuronidase (E7GGG.GUS) had been previously proven to provide partial protection from TC-1/A9-induced tumors, we also combined E7GGGHSP with E7GGG.GUS. The genes were inoculated either in mix in two gene gun shots or separately each gene in one shot into opposite sides of the abdomen. Neither mode of combined immunization induced higher protection than E7GGG.GUS alone. However, doubling the DNA dose considerably enhanced the antitumor efficacy of E7GGG.GUS.
  
  
  CONCLUSIONS
  We constructed highly immunogenic fusions of HPV16 E7 and E7GGG with mouse HSP70.1. Furthermore, we substantially enhanced protection against TC-1/A9 cells with downregulated MHC class I expression by doubling the pBSC/E7GGG.GUS dose, but we failed to demonstrate a beneficial effect of any combination of two fusion genes with different mechanisms causing enhancement of HPV16 E7 immunogenicity.
authors.affiliation: Institute of Hematology and Blood Transfusion, Department of Experimental Virology, U Nemocnice 1, 128 20 Prague 2, Czech Republic. danap@uhkt.cz
authors.forename: Dana
authors.initials: D
authors.lastname: Pokorná
authors.forename: Jana
authors.initials: J
authors.lastname: Macková
authors.forename: Martina
authors.initials: M
authors.lastname: Dusková
authors.forename: Simon
authors.initials: S
authors.lastname: Rittich
authors.forename: Viera
authors.initials: V
authors.lastname: Ludvíková
authors.forename: Michal
authors.initials: M
authors.lastname: Smahel
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology; D019470:Biolistics; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002472:Cell Transformation, Viral; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology; D007114:Immunization; D007279:Injections, Subcutaneous; D008175:Lung Neoplasms / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D041681:NIH 3T3 Cells; D009374:Neoplasms, Experimental / Q000517:prevention & control; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology*; D027383:Papillomaviridae / Q000235:genetics / Q000276:immunology*; D050725:Papillomavirus E7 Proteins; D010957:Plasmids; D013997:Time Factors; D014412:Tumor Virus Infections / Q000517:prevention & control; D014611:Vaccination; D019444:Vaccines, DNA / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D018840:HSP70 Heat-Shock Proteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D019444:Vaccines, DNA; C081884:heat-shock protein 70.1; C059731:oncogene protein E7, Human papillomavirus type 16