Combined immunization with fusion genes of mutated E7 gene of human papillomavirus type 16 did not enhance antitumor effect.

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. 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. 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.

Pokorná D ，Macková J ，Dusková M ，Rittich S ，Ludvíková V ，Smahel M ... -  《JOURNAL OF GENE MEDICINE》

Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli beta-glucuronidase.

Smahel M ，Pokorná D ，Macková J ，Vlasák J ... -  《JOURNAL OF GENE MEDICINE》

Modified HPV16 E7 Genes as DNA Vaccine against E7-Containing Oncogenic Cells.

Therapeutic vaccines against tumors associated with human papillomaviruses (HPV) should elicit cellular immune responses against early HPV antigens, primarily the oncoproteins E7 and E6. Because of safety concerns, the direct use of an unmodified oncogene is impossible in human DNA vaccination. Therefore, we introduced three point mutations into the pRb-binding site of HPV16 E7 oncogene to eliminate its transformation potential. The resultant gene was denoted E7GGG. The rates of expression and the cellular localization of E7 and E7GGG proteins were comparable. In immunization-challenge experiments, the efficacy of plasmids containing the E7, E7GGG, or fusion genes of HPV16 E7, viz. L1DeltaCE7(1-60) (M. Muller et al., 1997, Virology 234, 93-111), and Sig/E7/LAMP-1 (T. C. Wu et al., 1995, Proc. Natl. Acad. Sci. USA 92, 11671-11675), was compared. While tumors developed in all animals immunized with the wild-type E7 gene, a significant proportion of mice remained tumor-free after vaccination with the E7GGG gene. The fusion gene L1DeltaCE7(1-60) induced negligible protection, but Sig/E7/LAMP-1 conferred the highest protection. Intradermal immunization by gene gun proved superior to i.m. inoculation. In "therapeutic" experiments, a 1-day delay between inoculation of oncogenic cells and the start of DNA immunization resulted in partial therapeutic effect, but a 3-day delay produced a substantially lower immunization effect. A combination of Sig/E7/LAMP-1 and E7GGG genes did not enhance the immune response. These results demonstrate a significant enhancement of HPV16 E7 immunogenicity after mutagenesis of the pRb-binding site, but the mutated E7 gene did not excel the Sig/E7/LAMP-1 fusion gene.

Smahel M ，Síma P ，Ludvíková V ，Vonka V ... -  《VIROLOGY》

Enhancement of T cell-mediated and humoral immunity of beta-glucuronidase-based DNA vaccines against HPV16 E7 oncoprotein.

Smahel M ，Poláková I ，Pokorná D ，Ludvíková V ，Dusková M ，Vlasák J ... -  《INTERNATIONAL JOURNAL OF ONCOLOGY》

DNA vaccines based on chimeric potyvirus-like particles carrying HPV16 E7 peptide (aa 44-60).

Pokorna D ，Cerovska N ，Smahel M ，Moravec T ，Ludvikova V ，Mackova J ，Synkova H ，Duskova M ，Hozak P ，Veleminsky J ... -  《ONCOLOGY REPORTS》