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 CHIMERIC VIRUS PARTICLES AS IMMUNOGENS FOR INDUCING MOUSE AND HUMAN IMMUNE RESPONSES AGAINST HIV-1

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

* ENEA, Divisione Biotecnologie e Agricoltura. C.R. Casaccia. 00060 Rome, Italy

marusic_c@hotmail.com; benvenutoe@casaccia.enea.it

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

capone@iss.it

chimeric virus particles, dendritic cells, HIV-1, PVX, vaccine

Plants and plant viruses have recently been considered as attractive systems for expressing and delivering foreign proteins or peptides as immunogens to be used for the development of new vaccination strategies. 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. Plant virus coat proteins (CP) are particularly suitable carriers to present immunogenic peptides to the immune system. When properly fused at different positions on the capsid proteins, exogenous sequences are expressed in plant, originating recombinant viral coat proteins able to self-assemble and generate chimeric virus particles (CVPs) displaying the foreign sequence on their outer surface. The possibility to carry out a mucosal delivery of vaccine-expressing plants is particularly important for viruses transmitted mainly via mucosal surfaces such as human immunodeficiency virus type 1 (HIV-1). We assayed the immunogenicity of PVX-derived CVPs displaying the epitope ELDKWA (2F5e), located in the membrane proximal part of the gp41 ectodomain. We modified the PVX CP encoding gene by linking the sequence encoding the HIV-1 gp41-derived 2F5e and recombinant virus was used to infect Nicotiana benthamiana plants. Leaves from plants showing infection were collected, CVPs (PVX-2F5E) purified, and assessed by ELISA for the correct display of the HIV-1 epitope on their outer surface using the human mAb 2F5. Then, we immunized mice intranasally or intraperitoneally with purified CVPs. Sera from both i.n. or i.p. PVX-2F5E-immunized mice showed high levels of IgG specific for a synthetic peptide containing 2F5e. We investigated the human immune response to PVX-derived CVPs displaying the 2F5e in severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID). We provide evidence hu-PBL-SCID mice immunized with CVPs-pulsed autologous dendritic cells (DCs) are able to mount a specific human primary antibody response against the HIV-1-derived epitope. Remarkably, sera obtained from both normal and hu-PBL-SCID mice were endowed with anti-HIV-1 neutralizing activity. Here, we report also the use of PVX as vector to express the regulatory Tat protein in plant. Upon systemic infection of N.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 correct assembly of virions. In Western blot analysis of 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. The high molecular weight component was mainly in the membrane fractions. 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.