Transgenic chloroplasts are efficient sites for high‐yield production of the vaccinia virus envelope protein A27L in plant cells†

Abstract

SummaryOrthopoxviruses (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.

Expression of a Sheep Pox Virus Gene in Plant Systems under the Control of Plant Viral Regulatory Elements and with Sub-Cellular Targeting

“…Approximately the same level of VACV A27L protein production was achieved previously in plants transformed through the nuclear genome 24 .…”

Unsolved problems in plastid transformation

“…In agreement with the observed phenotype, analysis by immunofluorescence microscopy performed on protoplasts isolated from transformed or wild-type plants showed altered chlorophyll distribution. 23 Moreover, transmission electron microscopy (TEM) analyses of leaf tissues indicated an alteration of chloroplast membrane ultrastructure in transplastomic plants (Fig. 2).…”

“…3,[23][24][25] For example, we expressed in transplastomic tobacco plants the immunogenic proteins A27L of vaccinia virus that accumulated to about 18% of total soluble protein. 23 We observed that in transplastomic lines the accumulation of the Rubisco large and small subunits was reduced. In soil, transplastomic plants grew more slowly and were chlorotic.…”

“…23,28 Post-translational modifications, such as disulphide bond formations, can influence the stability of recombinant proteins produced in plastids contributing to protein accumulation. 28 Moreover, correct folding of the proteins by formation of disulphide bonds is often required for functional tertiary and quaternary structures and, therefore, for antigenicity of the recombinant proteins expressed in transplastomic plants.…”

Genetic Transformation in Tomato: Novel Tools to Improve Fruit Quality and Pharmaceutical Production

“…Major advantages of plant-made pharmaceuticals include low cost of production, higher scale-up capacity and lack of risk of contamination with mammalian pathogens. Several antigenic proteins have been produced in plant, examples are plant-made vaccines against smallpox, HIV and HPV (Human Papilloma Virus) (Lenzi et al, 2008;Rigano et al, 2009;Scotti et al, 2009). In addition, transgenic plants can represent a suitable vehicle for oral delivery of pharmaceuticals since the plant cell wall protects the recombinant antigen in the harsh condition of stomach and intestine (Sharma et al, 2008a).…”