Plantechno (Italy)

Plants have represent a promising alternative for biopharmaceutical proteins (Ma et al., 2003; Rossi et al., 2014). Many plant based edible vaccines have been shown to be effective in inducing local immune responses ( Rossi et al., 2013). Edible vaccines can activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining. This dual effect would provide first-line defense against pathogens invading through the mucosa. The antigens are released in the intestines are taken up by M cells that are present over the Payer’s patches (in the ileum) and the gut associated lymphoid tissue (GALT). Edible vaccines represent an important worldwide goal for the prevention of the enteric diseases, also in livestock. In particular, the enteric infections are a significant clinical problem in pigs. Verocytotoxic Escherichia (E.) coli strains are responsible for serious enterotoxaemia that causes important economic losses in the pig industry. The production of a vaccine for oral administration of transgenic seeds could be a practical and efficient system to prevent the infection and to reduce the antibiotic use. This study was focused on tobacco plants, previously transformed by agroinfection for the seed-specific expression of antigenic proteins (F18 adhesive fimbriae and the B subunit of the Vt2e toxin) as model of edible vaccines against verocytotoxic E. coli strains. The dietary administration of transgenic tobacco seeds promotes a significant increase in the number of mucosal IgA-producing cells of the tunica propria in both small and large intestine in mice ( Rossi et al., 2013). A protective effect of oral administration of transgenic tobacco seeds was also observed against verocytotoxic Escherichia coli infection in piglets (Rossi et al., 2014). The aim of this study was to assess the seed-expression stability, that is a important requirement in the vaccine production, of F 18 and Vt2e-B heterologous genes into the progeny of transformed tobacco plants.

Expression of the vhb gene, encoding the hemoglobin protein from Vitreoscilla spp. (VHb), has been shown to increase cell growth and protein synthesis, modify the oxygen-dependent product biosynthesis and the susceptibility to oxidative and nitrosative stresses in several host microrganisms, and to improve plant tolerance to flooding-submergence. A chimeric construct consisting of the CaMV35S promoter fused to the vhb gene and nopaline synthase terminator was transferred into white poplar (Populus alba L.) via Agrobacterium tumefaciens in order to test the generality of these phenomena. The presence of the vhb gene was demonstrated by Southern blot analysis. Accumulation of the vhb transcript and protein was detected in all the selected transgenic poplar lines. In vitro growth bioassays revealed that the vhb gene expression in transgenic poplar plants did not significantly affect their growth pattern. One out of the six selected transgenic lines showed significantly higher values for plant height and stem biomass in greenhouse conditions and exhibited enhancement of root biomass production and stem diameter when compared to the wild-type plants. However, no significant differences in chlorophyll a, b, total carotenoid and protein contents were observed. Two selected transgenic lines were characterized in more detail for tolerance to submergence, oxidative and nitrosative stresses. Under in vitro and in vivo submergence conditions, growth parameters and total protein content of transgenic VHb poplars were similar to those observed in the wild-type plants. In addition, leaf discs from the transgenic plants maintained in standard growth conditions did not reveal increased tolerance to oxidative stress by hydrogen peroxide compared to wild-type plants. In a parallel study, cell suspension cultures obtained from both wild-type and VHb transgenic lines were evaluated for growth and survival in the presence of oxidative and nitrosative stresses. No significant differences were observed between the tested VHb and wild-type poplar lines. Our results show that VHb expression in plants can have erratic effects since the enhancement of plant growth and biomass production and the tolerance to submergence, oxidative and nitrosative stresses are not consistently dependent on the presence of this specific function. Consequently, the genetic manipulation of plant oxygen metabolism must be carefully evaluated and extensive biochemical, molecular and cellular investigations are required to assess the real value of the final products.

Gaucher disease, the most common genetic lysosomal disorder, is caused by the lack of functional acid beta-glucosidase (GCase) and is currently treated at a very high cost by enzyme replacement therapy. In an attempt to provide a safe and cost-effective production system, human placental GCase was produced and purified from transgenic tobacco seeds. Plant-derived recombinant GCase was found to be enzymatically active, uptaken by human fibroblasts and free of immunogenic xylose and fucose residues. This report demonstrates the potential of plant bioreactors in the large-scale production of injectable proteins required for lifelong therapy.