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 - 3.25

POTATO VIRUS X CHIMERIC PARTICLES FOR VACCINE DEVELOPMENT

C. LICO*, C. MARUSIC**, F. CAPUANO*, P. RIZZA***, F. BELARDELLI***, E. BENVENUTO*, I. CAPONE***, S. BASCHIERI*

*) ENEA, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione di Genetica e Genomica Vegetale, C.R. Casaccia, Rome

**) ENEA, C.R. Trisaia, Rotondella (Mt), Italy

***) Istituto Superiore di Sanità, Laboratorio di Virologia, Rome, Italy

PVX, chimeric virus particle, vaccine, HIV-1 epitope

In the last decade the use of genetically modified plant viruses has been explored as efficient tool to express in plant tissues heterologous proteins of biomedical interest. A particular application of this technology is finalized to the production of innovative vaccines by generating chimeric virus particles (CVPs) expressing on their coat protein (CP) pathogen-derived epitopes (i.e. short aminoacid stretches known to be responsible of the activation of the immune response against a pathogen). In this case the viruses are not only a plant expression system but also fundamental vaccine components working as delivery tools.

We have previously demonstrated that the mucosal delivery of purified Potato Virus X (PVX) chimeric particles expressing the 2F5e epitope of HIV-1 gp160 protein, is able to induce strong IgA and IgG antibody responses in mouse models, without the need of adjuvants.

We are presently trying to improve this vaccine delivery system by modifying the CP of PVX to display longer immunostimulating epitopes.

Two modified viral expression vectors, derived from pPVX201, have been constructed to insert, as CP-fusion, HIV-1-derived epitopes of different lengths. The modified vector pPVXCC has been used to display epitopes able to induce neutralizing antibody-responses (8, 14 and 23 aminoacids) while the modified vector pPVXSMA has been used to display epitopes known to induce T-cell mediated immune responses (9 and 10 aminoacids).

The viral vectors (infectious transcripts) have been used to infect N. benthamiana plants. Ten days after infections RNA have been extracted from systemically infected leaves and reverse transcribed. Sequence analysis have been performed on PCR fragments amplified with PVX-specific primers.

Both pPVXCC and pPVXSMA are able to systemically infect plants, producing symptoms that are identical to those induced by pPVX201. Moreover, they both replicate stably in infected tissues, as assessed by repeated infection cycles with infected plant extracts.

The pPVXCC derived vectors carrying the sequence coding for 8 or 14 aa long epitopes (as opposite to the vector carrying the sequence coding for the 23 aa long epitope, which seems to be unable to start plant infection) generates CVPs able to systemically infect plants, without evident symptoms. Nonetheless both CVPs display correctly the exogenous sequences.

In the case of the pPVXSMA derived vectors, only the CVPs displaying the 9 aa epitope ends up in systemic infections, with symptoms undistinguishable from pPVXSMA and pPVX201, retaining the exogenous sequence.

From these results we can conclude that both modified vectors can be considered as interesting alternatives to pPVX201 for the production of CVPs displaying hexogenous sequences that are in the case of pPVXCC 14 aminoacid residues long and in the case of pPVXSMA 9 aminoacid residues long. However it is important to note that successful epitope display on hybrid CP is affected not only by the length of the sequence but also by its “quality” in terms of aminoacid composition.