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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 2.12

STABLE AND TRANSIENT EXPRESSION OF HIV-1 TAT PROTEIN IN PLANTS

LICO C.*, MARUSIC C.*, LOPEZ M.*, LATTANZI L.**, RIZZA P.**, BELARDELLI F.**, CAPONE I.**

*) ENEA, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, 00060 Rome, Italy

**) Istituto Superiore di Sanità, Laboratorio di Virologia, 00161 Rome, Italy

HIV-1, TAT, PVX, vaccine

During the last decade, commercial and academic interest in plants as heterologous expression system have significantly increased, especially for the production of biomedically important proteins. The employment of plants for the production of therapeutic proteins offers several advantages such as absence of mammalian pathogens, cost-effectiveness, large-scale production and relative ease in expression and purification. Both transgenic plants and plants infected with engineered plant viruses have been used in producing foreign proteins or peptides as immunogens to be used for the development of new vaccination strategies. Plants might also provide an ideal vehicle for oral delivery of vaccine antigens. The proof of this concept has been shown using several bacterial and viral proteins. The possibility to carry out a mucosal delivery of vaccine-expressing plants, potentially resulting also in the activation of the mucosal-associated immune system, is particularly important for viruses transmitted mainly via mucosal surfaces such as human immunodeficiency virus type 1 (HIV-1). Tat protein of HIV-1 is a small nuclear molecule of 14 kDa, warking as a transacting transcriptional activator, and is known to be involved in the beginning of transcription and in RNA chain elongation. Tat might be released from acute infected CD4+ T lymphocytes, and interacting with nearby activated uninfected T-cells, Tat it inhibits their proliferation. Tat is also immunogenic, and antibodies raised against it may have protective effects in controlling disease progression by inhibiting both the effect of extracellular Tat on HIV replication and its immunosuppressive effects on T cells. In addition, recent experiments showed that immunization with a biologically active wild-type Tat protein of HIV-1 elicits a broad immune response and controls infection in monkeys. These findings suggest that Tat, coupled with a vector delivering HIV-1 structural proteins, may be an important component of an effective multicomponent AIDS vaccine. For this reason we have expressed HIV-1 Tat protein into Lycopersicon esculentum using Agrobacterium tumefaciens-mediated gene transfer. We analysed expression levels of eleven independent transgenic tomato lines by semi-quantitative RT-PCR on RNA extracted from leaves. Three transformants showed high expression levels (12.9, 6.9 and 6.4 times more than a housekeeping gene used as a control). Protein expression analysis by Western blot revealed that Tat is present at detectable levels both in leaf extracts and in the fruit.

Moreover we report also the use of PVX viral vector as transient expression system for Tat protein.

Tat gene was cloned in the pPVX as single ORF, with the c-myc coding sequence at the 3' end. Upon systemic infection of Nicotiana benthamiana plants, the expression of foreign sequence has been verified by Western blot analysis. Our results have shown that the accessory ORF tat did not interfere with the correct assembly of PVX virions and in fractionated protein extracts, polyclonal anti-Tat antibodies recognised a faint band at 14 KDa (corresponding to monomeric Tat) and a stronger signal was detected at 48 KDa. We confirmed these results using monoclonal anti-c-myc antibodies. The high molecular weight component was mainly in the membrane fractions. We have estimated that the expression level in infected plant tissues is around 0,2% of the total proteins. Ongoing studies will evaluate if the immune response elicited by Tat protein expressed in plant tissues is comparable or better to that induced by E. coli-derived Tat protein.