THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2004; 6: 1092–1101. Published online 5 May 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.596 Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli β -glucuronidase Michal ˇ Smahel 1 * Dana Pokorn´ a 1 Jana Mackov´ a 1 Josef Vlas´ ak 2 1 Institute of Hematology and Blood Transfusion, Department of Experimental Virology, Prague, Czech Republic 2 Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, ˇ Cesk´ e Budˇ ejovice, Czech Republic *Correspondence to: Michal ˇ Smahel, Institute of Hematology and Blood Transfusion, Department of Experimental Virology, U Nemocnice 1, 128 20 Prague 2, Czech Republic. E-mail: smahel@uhkt.cz Received: 3 August 2003 Revised: 23 February 2004 Accepted: 2 March 2004 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. Methods and results To increase E7GGG immunogenicity we constructed fusion genes of E. coli β -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. Copyright  2004 John Wiley & Sons, Ltd. Keywords human papillomaviruses; E7 oncogene; GUS; DNA vaccine; fusion genes Introduction Administration of plasmid DNA is a simple vaccination approach that induces both humoral and cellular immune responses. Due to a number of advantages, DNA vaccines hold promise for future extensive use. They were demonstrated to activate effective immu- nity against both infectious agents and tumour cells. However, the Copyright  2004 John Wiley & Sons, Ltd.