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Plants have a demonstrated potential for large-scale, rapid production of recombinant proteins for diverse product applications, including subunit vaccines and monoclonal antibodies. In this field, the accent has recently shifted from the engineering of "edible" vaccines based on stable expression of target protein in transgenic or transplastomic plants to the development of purified formulated vaccines that are delivered via injection. The injectable vaccines are commonly produced using transient expression of target gene delivered into genetically unmodified plant host via viral or bacterial vectors. Most viral vectors are based on plant RNA viruses, where nonessential sequences are replaced with the gene of interest. Utilization of viral hybrids that consist of genes and regulatory elements of different virus species, or transcomplementation systems (vector/transgene) had a substantial impact on the level of target protein expression. Development and introduction of agroviral hybrid vectors that combine genetic elements of bacterial binary plasmids and plant viral vectors, and agroinfiltration as a tool of the vector delivery have resulted in significant progress in large-scale production of recombinant vaccines and monoclonal antibodies in plants. This article presents an overview of plant hybrid viral vector expression systems developed so far.

To co-express multiple target proteins, we engineered a single-component chimeric tobacco mosaic virus (TMV)-based vector containing homologous and heterologous capsid protein subgenomic RNA promoters. Delivery of this vector into Nicotiana benthamiana plants via agroinfiltration resulted in co-expression of two reporter genes within a single cell. Furthermore, co-expression of a host-specific antisense RNA or a silencing suppressor protein from this vector augmented the accumulation of green fluorescent protein or a vaccine antigen, hemagglutinin from avian influenza virus A/Vietnam/1194/04. These findings suggest that this chimeric vector utilizing the homologous and heterologous subgenomic TMV promoters has a potential for high-level production of multiple therapeutic proteins including monoclonal antibodies.

Expression of multiple therapeutic proteins from Tobacco mosaic virus (TMV)-based vectors was not successful when plants were coinoculated with a mixture of two TMV vectors engineered to express two foreign genes individually. Here, we have engineered and developed a defective RNA (dRNA)-based TMV vector (dRT-V) that utilizes two components of the same virus, with the dRNA component depending on the helper virus for replication. Agrobacterium-mediated coinoculation of Nicotiana benthamiana plants with both components of the dRT-V resulted in high-level expression of a human growth hormone and a lichenase-fused lethal factor protein of Bacillus anthracis. Furthermore, both heavy and light chains were expressed and assembled into a monoclonal antibody (mAb) specific to the protective antigen of B. anthracis, and the average yield of the purified antibody obtained was 120 mg/kg of fresh tissue. Our data suggest that dRT-V has a potential for rapid, cost-effective, large-scale manufacturing of multiple therapeutic proteins including mAbs in response to any biological emergencies., (Copyright 2010 Elsevier Inc. All rights reserved.)