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

PLANT-DERIVED ANTIGENS OF THE HUMAN PAPILLOMAVIRUS 16 (HPV16) FOR THE DEVELOPMENT OF NEW VACCINES AGAINST HPV16-ASSOCIATED CANCER

DIBELLO F.**, DI BONITO P.**., ACCARDI L.**, BITTI O.,* CIRILLI A.*, BENVENUTO E.*, GIORGI C.**, VENUTI A.*, 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

Human Papilloma Virus (HPV), PVX, vaccine

Plants are currently being used as cost-effective and safe heterologous expression system for the production of experimental vaccines. Transgenic plants can be used but, generally, the amount of foreign antigen is too low to be routinely used for practical purposes.

The use of recombinant plant viruses as vectors is an alternative, often resulting in the production of higher quantities of the protein of interest in a short time.

The human papilloma virus (HPV) is the primary causative agent in more than 95% of cervical cancer (second most common cause of cancer death in women worldwide), with HPV16 being the type most frequently found in these tumours. HPV16 is also involved in the pathogenesis of several other tumors. Vaccination against HPV has given promising results in animal models and clinical trials are currently being performed to assess the safety and immunogenicity of several vaccines (Breitburd & Coursaget, 1999).

Prophylactic vaccination essentially uses ‘virus-like particles’ (VLPs), self-assembled from the L1 major capsid protein in eukaryotic expression systems.

Therapeutic vaccination against HPV-associated tumors is currently achieved by using recombinant E7 protein. This oncoprotein is expressed at a high level in human cells during malignancy and hence is considered as 'tumor-associated antigen'.

In an attempt to develop both prophylactic and therapeutic new vaccines against HPV16, we tried to produce HPV16 L1 and E7 antigens in plants.

The L1 gene was firstly cloned into a potato virus X (PVX)-derived epichromosomal expression vector. When DNA plasmid was used to infect N. benthamiana plants, neither PVX symptoms nor recombinant L1 protein were detected. Most probably, the adjunctive HPV16 L1 gene interfered with the PVX replication process. Hence, for the production of this antigen, transgenic plants ('microtomato') have been produced. Data on the ongoing characterization of the transgenic lines will be presented.

Conversely, when a PVX-derived vector was used to express the original HPV16 E7 gene, high amounts of soluble E7 protein were detected.

The immunogenicity of the plant-derived E7 was analysed in a mouse model. PVX-E7-infected plant extracts (containing about 0.5 mg of E7 protein) were administered sub-cutaneously (four times, at 15 days-interval) with/without adjuvant. Fifteen days after the last booster, animals were bled and the sera were collected. ELISA test showed that the mice immunised with leaf extracts had developed anti-E7 antibodies. The “vaccinated” mice were subsequently challenged with tumor cells expressing E7. About 50% of mice immunised with PVX-E7 leaf extracts were protected. The addition of an adjuvant did not increase tumor protection. Cell-mediated immune response was also demonstrated.

Our results suggest that mice 'vaccinated' with the E7-containing plant extracts are able to develop an immune response effective to confer protection against tumour challenge. The fact that no purification and no adjuvant are needed, opens a very attractive way for the production of reliable and inexpensive therapeutic vaccine for HPV-associated cancer as well as for further insights into plant-derived adjuvants.

Breitburd F. & Coursaget P. (1999). ‘Human papillomavirus vaccines’. Semin. Cancer Biol. 9, 431-444.