Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 3.24

PLANT-DERIVED SEQUENCES AND PLANT VIRAL GENES FUSED WITH A MODIFIED HPV16 E7 GENE FOR THE DEVELOPMENT OF NEW DNA THERAPEUTIC VACCINES AGAINST HPV-RELATED TUMORS

S. MASSA*, P. SIMEONE**, E. ILLIANO*, E. BENVENUTO*, A. VENUTI**, R. FRANCONI*

*) ENEA, BIOTEC GEN, CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy

**) Istituti Fisioterapici Ospitalieri (IFO) - Istituto Regina Elena per lo Studio e la Cura dei Tumori, Via delle Messi d'Oro 156, 00158 Rome, Italy

Human Papilloma Virus 16, E7 oncoprotein, Potato Virus X, coat protein

The high-risk types of Human Papillomavirus (HPV16, 18) are currently accepted as the primary etiologic agents of cervical cancer. Being the HPV16 E7 oncoprotein constitutively expressed in these tumor cells and responsible for the onset and maintenance of the transformed state, it represents a promising target for the development of therapeutic vaccines against HPV-associated malignancies.

DNA vaccination is an attractive approach for HPV immunotherapy and, in pre-clinical studies, it has shown to elicit long-term humoral and cellular immunity. Clinical trials are also in progress.

Besides their exploitation as biofactories, plants and plant viruses can offer new opportunities to increase the efficacy of DNA vaccines by providing sequences that are primary antigen in humans.

In this perspective, we firstly introduced three point mutations into the retinoblastoma (pRb)-binding site of HPV16 E7 oncogene to abolish its transformation potential (“E7GGG” gene). Then we made N- and C-terminal gene fusion of the E7GGG gene with, respectively, the Potato Virus X coat protein (PVX-CP) and the signal peptide coding sequence of the polygalacturonase-inhibiting protein (PGIP) from bean.

The former gene fusion should exhibit high immunogenicity. In fact, it has already been shown that such an approach with other tumor associated antigens, leads to highly aggregated, self-assembling structures able to increase the CD4⁺ immune response¹. The E7GGG sequence from HPV16 was directly assembled with the PVX-CP or fused in frame to the fourth codon of PVX-CP via the aminoacid linker ”Gly-Pro-Gly-Pro”.

We already reported the enhanced expression level of a secretory HPV16 E7 protein (PGIP-E7) in N. benthamiana plants. Fusion to the PGIP sequence in a DNA vaccine, should improve E7 protein expression level and “visibility” by the immune system.

The synthetic genes were cloned into a mammalian expression vector. The above mentioned DNA constructs are currently being tested in order to assess whether they induce humoral and/or cellular immune responses and protection from cancer in vaccinated mice challenged with a tumorigenic dose of E7-expressing singeneic tumor cells. Data on biological effects will be presented.

¹. Savelyeva N. et al. (2001) 'Plant viral genes in DNA idiotypic vaccines activate linked CD4⁺ T-cell mediated immunity against B-cell malignancies'. Nature Biotechnology 19: 760-764.