Vaccine 28 (2010) 1506–1513 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine DNA vaccine against human papillomavirus type 16: Modifications of the E6 oncogene Ingrid Poláková ∗ , Dana Pokorná, Martina Duˇ sková, Michal ˇ Smahel Institute of Hematology and Blood Transfusion, Department of Experimental Virology, U Nemocnice 1, 12820 Prague 2, Czech Republic article info Article history: Received 23 October 2009 Received in revised form 16 November 2009 Accepted 21 November 2009 Available online 8 December 2009 Keywords: Human papillomavirus E6 oncogene DNA vaccine Gene gun Immunogenicity abstract Since its discovery, DNA vaccination has become an effective strategy for the development of vaccines against cancer including cervical carcinoma (CC). The formation of CC is associated with human papillo- mavirus (HPV) infection. Viral E6 and E7 oncoproteins are suitable targets for therapeutic vaccination. To adapt the HPV16 E6 oncogene for DNA immunisation, we performed several modifications. First we fused the E6 gene with the 5 ′ or 3 ′ -terminus of the Escherichia coli -glucuronidase (GUS) gene and showed enhanced immunogenicity of the 3 ′ fusion (GUS.E6). Then, as the E6 oncogene contains two alternative introns that result in the production of truncated forms of the E6 protein, we abolished the 5 ′ splice site in the E6 gene. This modification completely eliminated the expression of the truncated E6 transcripts and thus increased the production of the full-length E6 protein. At the same time, it moderately reduced the immunogenicity of the modified non-fused (E6cc) or fused (GUS.E6cc) genes, probably as a consequence of the substitution in the immunodominant E6 epitope following the abolishment of the splice site. Fur- thermore, we reduced the oncogenicity of the E6 protein by two point mutations (E6GT) that, together, prevented E6-mediated p53 degradation. Finally, we constructed the GUS.E6GT gene characterized by enhanced safety and immunogenicity when compared with the wild-type E6 gene. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Persistent infection with human papillomaviruses (HPV) is the main etiological factor in cervical cancer, the second most common cancer in women worldwide. Oncogenic high-risk (HR) HPV geno- types 16 and 18 are responsible for approximately 70% of all cervical cancers. Recently, two prophylactic vaccines based on virus-like particles (VLPs) produced by recombinant technology and protect- ing against infection with HPV16 and HPV18 have been licensed [1]. However, the development of therapeutic vaccines is still a top- ical problem, as preventive vaccination is of limited use and cannot cope with current HPV infection [2]. Since the viral oncoproteins E6 and E7 that are constitutively produced in all HPV-infected cells and that contribute to the transformation of epithelial skin or mucosal cells are also necessary for the maintenance of the transformed state [3], they became promising targets for the development of the therapeutic HPV vaccines. DNA vaccines represent a potential form of antigen-specific immunotherapy of tumours because they can induce cytotoxic T- lymphocyte (CTL) response [4]. However, the low efficacy of DNA immunisation hampered its clinical use. Several strategies enhanc- ing immunogenicity of the DNA vaccines have been developed ∗ Corresponding author. Tel.: +420 221 977 302; fax: +420 221 977 392. E-mail address: ingrid.polakova@uhkt.cz (I. Poláková). including the modification of an antigen-encoding gene [5]. For clinical use of DNA vaccines, their safety must also be carefully considered. In our previous studies, we focused on the modifi- cation of the HPV16 E7 oncogene. To reduce its transformation potential, we altered it by point mutations resulting in the sub- stitution of three amino acids in the pRb-binding site of the E7 protein [6]. Furthermore, to enhance its immunogenicity, we fused the modified E7GGG gene with sequences encoding sorting signals of lysosome-associated membrane protein 1 (LAMP-1), Escherichia coli -glucuronidase (GUS) or mouse heat shock protein 70 (Hsp70) and demonstrated a superior antitumour effect of the E7GGG.GUS chimeric construct [7–9]. As the E7 oncoprotein is a relatively small protein (98 amino acids) with a limited number of epitopes [10] and immunity against the E6 oncoprotein (158 amino acids) is more readily induced in HPV16-infected people and is probably more important for the elimination of infected cells [11,12], E6 should also be included in the therapeutic HPV vaccines. The HPV16 E6 oncoprotein is a multifunctional protein with several cellular targets. The first identified target, and apparently the most relevant, is the p53 tumour suppressor protein that can promote cell cycle arrest or apoptosis in infected cells. To over- come this obstacle, the E6 protein abrogates the functions of the p53 protein by inducing its degradation through the ubiquitin- proteasome pathway. The cellular E6AP ubiquitin ligase that binds both E6 and p53 plays a critical role in this process. Furthermore, the E6 oncoprotein can inhibit p53 activity independently of inducing 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.11.069