title: Transgenic chloroplasts are efficient sites for high-yield production of the vaccinia virus envelope protein A27L in plant cellsdagger.
abstract: Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost-effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein-based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500-fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast-synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.
authors.affiliation: Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples 'Federico II', Via Università, Portici, Italy.
authors.forename: M Manuela
authors.initials: MM
authors.lastname: Rigano
authors.forename: Carmela
authors.initials: C
authors.lastname: Manna
authors.forename: Anna
authors.initials: A
authors.lastname: Giulini
authors.forename: Emanuela
authors.initials: E
authors.lastname: Pedrazzini
authors.forename: Maria
authors.initials: M
authors.lastname: Capobianchi
authors.forename: Concetta
authors.initials: C
authors.lastname: Castilletti
authors.forename: Antonino
authors.initials: A
authors.lastname: Di Caro
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ippolito
authors.forename: Paola
authors.initials: P
authors.lastname: Beggio
authors.forename: Carlo
authors.initials: C
authors.lastname: De Giuli Morghen
authors.forename: Luigi
authors.initials: L
authors.lastname: Monti
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vitale
authors.forename: Teodoro
authors.initials: T
authors.lastname: Cardi
mesh_terms: D002736:Chloroplasts / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D054628:Genome, Chloroplast; D030821:Plants, Genetically Modified / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011523:Protoplasts / Q000276:immunology / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014170:Transformation, Genetic; D019076:Transgenes; D014616:Vaccinia virus / Q000235:genetics* / Q000276:immunology; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: C110360:A27L protein, Orthopoxvirus; D018749:RNA, Plant; D011994:Recombinant Proteins; D014764:Viral Proteins