A launch vector for the production of vaccine antigens in plants

Abstract

Historically, most vaccines have been based on killed or live‐attenuated infectious agents. Although very successful at immunizing populations against disease, both approaches raise safety concerns and often have limited production capacity. This has resulted in increased emphasis on the development of subunit vaccines. Several recombinant systems have been considered for subunit vaccine manufacture, including plants, which offer advantages both in cost and in scale of production. We have developed a plant expression system utilizing a ‘launch vector’, which combines the advantageous features of standard agrobacterial binary plasmids and plant viral vectors, to achieve high‐level target antigen expression in plants. As an additional feature, to aid in target expression, stability and purification, we have engineered a thermostable carrier molecule to which antigens are fused. We have applied this launch vector/carrier system to engineer and express target antigens from various pathogens, including, influenza A/Vietnam/04 (H5N1) virus.

Optimization and Utilization of <em>Agrobacterium</em>-mediated Transient Protein Production in <em>Nicotiana</em>

“…The remaining 5-10% of leaf area was not infiltrated because of some floating of leaves on the surface of the Agrobacterium suspension during application of the vacuum. Since the launch vector has ability for cell-to-cell movement 18 , transient protein accumulation occurs in entire leaves as well as petioles at 7 dpi. At 10 dpi, the estimated GFP production was slightly lower because the pBID4 expression vector is able to move from cell to cell but not to move systemically 18 ; therefore, newly grown leaves do not contain the vector and do not contribute to production of target.…”

“…Since the launch vector has ability for cell-to-cell movement 18 , transient protein accumulation occurs in entire leaves as well as petioles at 7 dpi. At 10 dpi, the estimated GFP production was slightly lower because the pBID4 expression vector is able to move from cell to cell but not to move systemically 18 ; therefore, newly grown leaves do not contain the vector and do not contribute to production of target. In addition, degradation of the recombinant protein over time may contribute to reduced protein level at 10 dpi.…”

“…These strains, isolated from the crown-gall of natural hosts, were kindly provided by Dr. Gelvin (Purdue University, West Lafayette, Indiana). To examine their utility in transient protein production, we infiltrated N. benthamiana with the following strains carrying pBID4-LicKM 18 : A. rhizogenes (A4) and A. tumefaciens wild-type Nester strains A348, A208, and A281 (named At6, At10, and At77, respectively), as well as engineered laboratory strains of A. tumefaciens GV3101, C58C1, and LBA4404. The infiltrated leaves were collected at 7 dpi and the level of LicKM expression was estimated by Western blot assay.…”

“…Transient protein production offers several advantages over production in transgenic plants, including short timeframe to achieve expression and accumulation 16 , and can be achieved by introducing bacterial binary vectors or recombinant plant viral vectors into plant tissues 4 . The most advanced transient expression system is based on the use of 'launch vectors' that combine components of plant viruses and binary plasmids, and are delivered by agroinfiltration 17,18 . Agroinfiltration of a launch vector based on Tobacco mosaic virus (TMV) has been successfully applied at lab scale to produce vaccine antigens against pathogens such as human papilloma virus 19 , Yersinia pestis 20 , influenza viruses A 21,22 , Bacillus anthracis 23 , and smallpox virus 24 in N. benthamiana leaves.…”

Expression, purification, and characterization of theNecator americanusaspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine

“…The synthesized sequence was cloned into the Tobacco Mosaic virus (TMV)-based launch expression vector pGRD4 23,24 with a PR-1a signal peptide of Nicotiana tabacum (BAA14220) replacing the native Na-APR-1 signal peptide at the N-terminus. 25 A His tag (6xHIS), as well as ER retention sequence (KDEL), was inserted at the C-terminus (Fig.…”