Malaria Epitopes Expressed on the surface of Recombinant Tobacco Mosaic Virus

Abstract

Using malaria as a model disease, we engineered the surface of tobacco mosaic tobamovirus (TMV) for presentation of selected epitopes to the mammalian immune system. The TMV coat protein is a well-characterized and abundant self-assembling polymer previously shown to be a highly immunogenic carrier. Selected B-cell epitopes were either inserted into the surface loop region of the TMV coat protein or fused to the C terminus using the leaky stop signal derived from the replicase protein reading frame. Tobacco plants systemically infected with each of these constructs contained high titers of genetically stable recombinant virus, enabling purification of the chimeric particles in high yield. Symptoms induced in tobacco ranged from a normal mosaic pattern similar to that induced by the parental U1 strain to a unique bright yellow mosaic. As measured by quantitative ELISA against synthetic peptide standards, wild type TMV coat protein and fusion protein synthesized by the leaky stop mechanism coassembled into virus particles at the predicted ratio of approximately 20:1. Recombinant plant viruses have the potential to meet the need for scalable and cost effective production of subunit vaccines that can be easily stored and administered.

Replication of tobamovirus RNA

“…The resulting virus multiplies systemically in host plants, and the wild-type CP and fusion protein co-assemble into virus particles. 94) Using this vector, the angiotensin I-converting enzyme inhibitor peptide, 94) epitopes from the influenza virus hemagglutinin and human immunodeficiency virus type I envelope protein, 95) and the malarial epitope, 90) have been displayed successfully on the surfaces of recombinant tobamovirus particles. In addition to the production of foreign proteins or peptides, pioneering work has been performed with tobamovirus vectors with respect to demonstrating virus-induced gene silencing 96) and to engineering plant metabolic pathways for the production of novel compounds via epigenetic expression of foreign genes.…”

Tobacco mosaic virus and the virescence of biotechnology

“…The principle that chimeric virus particles had potential as protective immunogens was thus established with TMV, as well as with the more commonly known hepatitis B surface antigen particles (Valenzuela et al 1985). After it became possible to synthesize infectious RNA from cloned cDNA copies of plant viral genomes, chimeric virus particles derived from TMV and other viruses were shown to present foreign peptides on their surface without signi¢cantly inhibiting the ability of the viruses to replicate and to infect whole plants systemically (Porta et al 1994;Turpen et al 1995;Fitchen et al 1995).…”

Impfschutz durch den Verzehr von gentechnisch verändertem Obst oder Gemüse?

“…[7] 1995 HIV, Typ 1 chim~ires Hª des cowpea Vigna unguiculata (i) mosaic virus mit Epitopbereich des HIV-1 [8] 1995 Plasmodium malariae chim~ires Hª des tobacco mosaic virus mit Epitopbereich von P. malariae [9] 1995 E. coli Untereinheit (11,6 kDa) des Hitze-Nicotiana tabacum (t) instabilen Enterotoxins von Solanum tuberosum (t) E. coli [10] 1995 Hepatitis B Virus Hª des Hepatitis B Nicotiana tabacum (t) Virus [11] 1996 Norwalk Virus virales Kapsid-Protein (58 kDa) Nicotiana tabacurn (t) des Norwalk Virus [12] Solanum tuberosum (t) Untersuchungen aus den Jahren 1990 bis 1994 haben gezeigt, dat~ in Analogie zu der Synthese und Assemblierung ron Antik6rpern in tierischen Zellen auch in pflanzlichen ZeUen die Untereinheiten der Antik6rper zun~ichst als Precursor mit Signalsequenzen im Zytoplasma synthetisiert und nach Prozessierung ira ER unter Mithilfe ron Chaperonen assembliert werden [16][17][18][19]. Darª hinaus konnte gezeigt werden, dafl das pflanzliche System auch zur Synthese von sekretorischen Antik6rpern bef~.higt ist [15], wie sie unter anderem auch im Darmtrakt auftreten.…”