Generation of multi-layered protein bodies in N. benthamiana for the encapsulation of vaccine antigens.

The ability of plants to assemble particulate structures such as virus-like particles and protein storage organelles allows the direct bioencapsulation of recombinant proteins during the manufacturing process, which holds promise for the development of new drug delivery vehicles. Storage organelles found in plants such as protein bodies (PBs) have been successfully used as tools for accumulation and encapsulation of recombinant proteins. The fusion of sequences derived from 27-kDa-γ-zein, a major storage protein of maize, with a protein of interest leads to the incorporation of the chimeric protein into the stable and protected environment inside newly induced PBs. While this procedure has proven successful for several, but not all recombinant proteins, the aim of this study was to refine the technology by using a combination of PB-forming proteins, thereby generating multi-layered protein assemblies in N. benthamiana. We used fluorescent proteins to demonstrate that up to three proteinaceous components can be incorporated into different layers. In addition to 27-kDa-γ-zein, which is essential for PB initiation, 16-kDa-γ-zein was identified as a key element to promote the incorporation of a third zein-component into the core of the PBs. We show that a vaccine antigen could be incorporated into the matrix of multi-layered PBs, and the protein microparticles were characterized by confocal and electron microscopy as well as flow cytometry. In future, this approach will enable the generation of designer PBs that serve as drug carriers and integrate multiple components that can be functionalized in different ways.

Plant-derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells.

The encapsulation of biopharmaceuticals into micro- or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N-terminal sequence of the maize storage protein, γ-zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N-terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration-based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen-presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.

Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum: implications for prolamin evolution.

In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD γ-zein locates at the interface between a core of alcohol-soluble α-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD γ-zein. 16-kD γ-zein originates from 27-kD γ-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD γ-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD γ-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two γ-zeins indicates that the N-terminal region of 16-kD γ-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs.

Plant Molecular Farming: Much More than Medicines.

Plants have emerged as commercially relevant production systems for pharmaceutical and nonpharmaceutical products. Currently, the commercially available nonpharmaceutical products outnumber the medical products of plant molecular farming, reflecting the shorter development times and lower regulatory burden of the former. Nonpharmaceutical products benefit more from the low costs and greater scalability of plant production systems without incurring the high costs associated with downstream processing and purification of pharmaceuticals. In this review, we explore the areas where plant-based manufacturing can make the greatest impact, focusing on commercialized products such as antibodies, enzymes, and growth factors that are used as research-grade or diagnostic reagents, cosmetic ingredients, and biosensors or biocatalysts. An outlook is provided on high-volume, low-margin proteins such as industrial enzymes that can be applied as crude extracts or unprocessed plant tissues in the feed, biofuel, and papermaking industries.

The Encapsulation of Hemagglutinin in Protein Bodies Achieves a Stronger Immune Response in Mice than the Soluble Antigen.

Zein is a water-insoluble polymer from maize seeds that has been widely used to produce carrier particles for the delivery of therapeutic molecules. We encapsulated a recombinant model vaccine antigen in newly formed zein bodies in planta by generating a fusion construct comprising the ectodomain of hemagglutinin subtype 5 and the N-terminal part of γ-zein. The chimeric protein was transiently produced in tobacco leaves, and H5-containing protein bodies (PBs) were used to immunize mice. An immune response was achieved in all mice treated with H5-zein, even at low doses. The fusion to zein markedly enhanced the IgG response compared the soluble H5 control, and the effect was similar to a commercial adjuvant. The co-administration of adjuvants with the H5-zein bodies did not enhance the immune response any further, suggesting that the zein portion itself mediates an adjuvant effect. While the zein portion used to induce protein body formation was only weakly immunogenic, our results indicate that zein-induced PBs are promising production and delivery vehicles for subunit vaccines.

Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants.

The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans.