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

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Oral Communication Abstract - S2f

IMMUNOMODULATION OF CMV (CUCUMBER MOSAIC VIRUS) IN TOMATO BY INTRACELLULAR SINGLE CHAIN ANTIBODY FRAGMENTS

M. E. VILLANI, E. BENVENUTO, R. FRANCONI

ENEA, BIOTEC GEN, CR Casaccia, Via Anguillarese 301, 00060 Roma

ntrabody, scFv, immunotherapy, CMV

Immunomodulation by using recombinant antibodies is an emerging tool for manipulating animal as well as plant metabolism.

In order to isolate recombinant single-chain variable fragment (scFv) antibodies specific for the cucumber mosaic virus (CMV), a pandemic plant pathogen, we used a phage display library composed of intrinsically stable scFvs, able to correctly fold in the reducing environment of the cytoplasm ('F8 library'). Several phage clones were obtained after four rounds of 'biopanning’ on the immobilised virus. Two clones were analysed in details. Affinity measurements revealed affinities for CMV in the range of nanomolarity, suggesting that these scFv fragments could represent a valuable tool for inexpensive diagnosis and potential inhibitors of virus multiplication.

In order to protect the plants from virus infection, we constructed transgenic microtomato plants expressing the scFv in the cytoplasm, the place in which virus replication occurs. Several transgenic lines were selected for scFv protein expression. Challenge of the T₁ generations, performed utilizing CMV-infected N. benthamiana plant extracts, led to the identification of at least two transgenic lines protected against CMV infection. The T₂ generation of these plants was challenged using a viral load extimated to be in the range of 20 µg/ml. After 15 days post inoculation, these plants showed no evident systemic symptoms, while wild-type microtomatoes were all severe affected. Among the transgenic plants, some of them showed delayed mild symptoms after 1 months p.i. Analysis of scFv expression revealed a correlation between the level of resistance and the quantity of the protein.

These data represent the first demonstration that recombinant antibodies derived from the 'F8 library' can act as effective intrabodies in vivo.