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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

PVX-MEDIATED EXPRESSION OF THE E7 ONCOPROTEIN DERIVED FROM THE HUMAN PAPILLOMAVIRUS 16: EFFECTS OF N- AND C-TERMINAL MODIFICATIONS AND OF DIFFERENT PLANT HOSTS SYSTEMS

DIBELLO F.**, GOBBI C.*, CENTENO MARTIN M. L.****, BITTI O.*, VENUTI A.***, GIORGI C.**, FRANCONI R.*

* ENEA, Casaccia, BIOAG BIMO, Roma

franconi@casaccia.enea.it

** Istituto Superiore di Sanità, Roma

*** Istituti Fisioterapici Ospitalieri (IFO), Istituto Regina Elena Studio e Cura dei Tumori, Roma

**** Università di Santiago de Compostela, Spagna

PVX, vaccine, HPV16

Plants represent effective tools for antigen production and delivery. The ectopic expression of heterologous proteins through viral vectors avoids some of the inherent limitations of stable expression derived from transformation procedures (low expression levels and time-consuming regeneration of plants).

We had previously demonstrated that, by using the potato virus X (PVX) as a vector, it is possible to quickly assess the compartmentalisation of a single-chain Fv antibody (scFv) inside the plant cell (Franconi et al., 1999). Moreover, scFv protein expression levels may significantly vary in different plant hosts and tissues (Roggero et al., 2001).

Object of the present study is the E7 oncoprotein derived from the human papilloma virus type 16 (HPV16), which is expressed at high levels in cervical cancer and, for this, is considered as ‘tumor associated antigen’. Therefore it represents a good candidate for the development of a plant-derived therapeutic vaccine against tumor.

We have recently demonstrated that plant extracts containing HPV16 E7 protein act as effective immunogen, able to confer protection against tumor challenge in a mouse model system.

In an attempt to improve expression levels and simplify protein purification procedures, various constructs of the E7 gene were made, with or without an N-terminal signal sequence for secretion. The signal peptide coding sequence of a bean protein (polygalacturonase-inhibiting protein, PGIP) was fused to the E7 gene and then cloned in a PVX-derived vector.

Both cytoplasmic and secretory genes were subsequently endowed of C-terminal ‘tags', such as the His- tag and the Flag-tag.

N. benthamiana plants were mechanically infected with each different construct and symptoms were observed with all of them. ELISA and Western blot analysis carried out on the symptomatic leaves confirmed the presence of the E7 protein. Interestingly, the PGIP-E7 fusions showed higher expression levels, possibly due to a stabilisation effect on the protein. Checking for the presence of the E7 protein in the apoplast and establishing proper purification protocols are currently being performed.

We also investigated E7 protein expression in different PVX hosts like Nicotiana rustica, Nicotiana tabacum, Lycopersicon esculentum and Chenopodium quinoa. Results show that each species behaves differently.

The optimisation of the plant species to be used (in terms of expression levels, absence of toxic compounds, maintenance of biological activity, etc.) represents an important aspect in order to ameliorate purification and vaccination protocols.

Franconi R, et al. (1999) 'Functional expression in bacteria and in plants of an scFv antibody fragment against tospoviruses'. Immunotechnology 4, 189-201.

Roggero P., et al. (2001) 'The expression of a single-chain Fv antibody fragment in different plant hosts and tissues by using potato virus X as a vector'. Protein Expression and Purification 22, 70-74.