Plant-based vaccines for animal health.

Plant-based vaccines are recombinant protein subunit vaccines. Ideally, the choice of plant species used to produce the selected antigen should allow for oral delivery in the form of an edible vaccine. These vaccines are well suited to combat diseases where there is a clear antigen candidate, and where the costs of production or delivery for any current vaccine are prohibitive. Several academic and industrial research groups are currently investigating the use of plant-based vaccines in both humans and animals. To date, the most advanced human vaccine projects have successfully completed phase I clinical trials, and animal vaccine projects have given promising data in early phase trials targeting specific animal species. In this article the advantages offered by plant-based vaccines will be presented, progress on the most advanced vaccine candidates will be summarised, and the path ahead will be outlined. Although the focus of this paper is on the application of plant-based vaccines in the field of animal health, principally their use in domestic livestock, examples of the use of plant-based vaccines in the field of human health will also be discussed.

Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants.

The use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. As the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. Currently, the cost of biopharmaceuticals limits their availability. Plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. Here, we discuss recent developments in this field and possible environmental concerns.

Plant-based vaccines: unique advantages.

Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

Disruption of the 3' phosphoglycerate kinase (pgk) gene in Aspergillus nidulans.

We report the isolation of a pgk- mutant strain of Aspergillus nidulans by means of a gene disruption strategy, and demonstrate that the pgk gene is located on chromosome VIII. The pgk- mutant conidiates poorly, will only grow on media supplemented with both a glycolytic and a gluconeogenic carbon source, and is inhibited by hexoses.

Intermolecular interactions between the A and B subunits of heat-labile enterotoxin from Escherichia coli promote holotoxin assembly and stability in vivo.

Cholera toxin and the related heat-labile enterotoxin (LT) produced by Escherichia coli consist of a holotoxin of one A subunit and five B subunits (AB5). Here we investigate the domains of the A subunit (EtxA) of E. coli LT which influence the events of B-subunit (EtxB) oligomerization and the formation of a stable AB5 holotoxin complex. We show that the C-terminal 14 amino acids of the A subunit comprise two functional domains that differentially affect oligomerization and holotoxin stability. Deletion of the last 14 amino acids (-14) from the A subunit resulted in a molecule that was significantly impaired in its capacity to promote the assembly of a mutant B subunit, EtxB191.5. In contrast, deletion of the last four amino acids (-4) from the A subunit gave a molecule that retained such a capacity. This suggests that C-terminal residues within the -14 to -4 region of the A subunit are important for promoting the oligomerization of EtxB. In addition, we demonstrate that the truncated A subunit lacking the last 4 amino acids was unable to form a stable AB5 holotoxin complex even though it promoted B-subunit oligomerization. This suggests that the last 4 residues of the A subunit function as an "anchoring" sequence responsible for maintaining the stability of A/B subunit interaction during holotoxin assembly. These data represent an important example of how intermolecular interactions between polypeptides in vivo can modulate the folding and assembly of a macromolecular complex.

Functional analysis of the expression of the 3'-phosphoglycerate kinase pgk gene in Aspergillus nidulans.

A functional analysis of the Aspergillus nidulans 3-phosphoglycerate kinase pgk promoter was undertaken using gene fusions to the lacZ gene of Escherichia coli, and introducing these into a beta-galactosidase-deficient strain of A. nidulans. Expression of a particular gene fusion in transformed strains depends upon the site of integration of the vector into the genome, and when specifically targeted to the catabolic quinate dehydrogenase qutE (selective marker) locus is directly proportional to its copy number. The analysis of transformed strains with single copies of pgk promoter deletion--lacZ fusions at the qutE locus identified three constitutive, positively acting sequence elements in the pgk gene. Sequence located between -161 and -120 nucleotides relative to the transcript start site +1, and including an element with a seven-out-of-eight nucleotide match (AAGCAAAT; -131 to -124) to the consensus eukaryotic octamer sequence ATGCAAAT, is essential for expression, and deletion of the complete 41-nucleotide sequence abolishes transcription. Sequence encompassing codons 14 to 183 and including the two introns of pgk contributes approximately one-third of the total activity, and far upstream sequence 5' to position -638 contributes approximately a further one-third total activity. In addition, sequence located -638 to -488 nucleotides, which includes an apparent consensus feature of A. nidulans glycolytic genes, affects carbon source-dependent regulation of expression. This region is required for an approximately 50% increase in pgk expression when A. nidulans is grown on gluconeogenic compared with glycolytic carbon sources.