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

Orthopoxviruses (OPV) have recently received increased attention, due to their potential use in bioterrorism and to 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 VACV protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to ~ 1.7 mg/g fresh weight). This level of A27L accumulation is 500-fold higher than the one in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants had 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 the serum from a patient recently infected by a zoonotic orthopoxvirus. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.