SummaryThe 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV ), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant).Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg /g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg /g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and G M1 -ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (∼100 mg for a dose of 500 µg antigen) and would permit encapsulation of freeze-dried material or pill formation.

The 2L21 epitope of the VP2 protein from the canine parvovirus (CPV), fused to the cholera toxin B subunit (CTB-2L21), was expressed in transgenic tobacco chloroplasts. Mice and rabbits that received protein-enriched leaf extracts by parenteral route produced high titers of anti-2L21 antibodies able to recognize the VP2 protein. Rabbit sera were able to neutralize CPV in an in vitro infection assay with an efficacy similar to the anti-2L21 neutralizing monoclonal antibody 3C9. Anti-2L21 IgG and seric IgA antibodies were elicited when mice were gavaged with a suspension of pulverized tissues from CTB-2L21 transformed plants. Combined immunization (a single parenteral injection followed by oral boosters) shows that oral boosters help to maintain the anti-2L21 IgG response induced after a single injection, whereas parenteral administration of the antigen primes the subsequent oral boosters by promoting the induction of anti-2L21 seric IgA antibodies. Despite the induced humoral response, antibodies elicited by oral delivery did not show neutralizing capacity in the in vitro assay. The high yield of the fusion protein permits the preparation of a high number of vaccine doses from a single plant and makes feasible the oral vaccination using a small amount of crude plant material. However, a big effort has still to be done to enhance the protective efficacy of subunit vaccines by the oral route.

Cultivated potato crops are sensitive to drought stress, reducing yield and tuber quality when the soil water potential drops to -0.3 MPa. However, drought not only affects plant growth and physiological activity, but this stress also induces biochemical and molecular changes in the cellular gene expression profile, triggering genes that play a direct role in plant protection and gene regulation. The genes involved in regulation include C-repeat Binding Factors/Dehydration responsive element binding (CBF/DREB) transcription factors, which increase cold, drought and salt tolerance in different plant species. The aim of this research was to evaluate whether the overexpression of the ScCBF1 gene from Solanum commersonii enhances drought stress tolerance in transgenic Solanum tuberosum and S. commersonii plantlets grown in vitro and induces drought adaptation mechanisms, such as osmoprotectors and genes for osmotic adjustment and membrane stability. Drought conditions were simulated through the addition of polyethylene glycol (PEG4000) to hormone-free MS medium. The vegetative growth, root development, proline content, and ScCBF1, Δ1-pyrroline-5-carboxylate synthetase (P5CS) and Dehydrins like genes (DNH10) expression were evaluated. The constitutive in vitro overexpression of ScCBF1 in both potato species showed better overall plantlet growth and root development under drought stress and higher proline levels in the stems and leaves. A significant increase in DNH10 expression was also associated with drought stress. In summary, the expression of ScCBF1 in potatoes induces responses associated with drought adaptation mechanisms, resulting in better overall plant growth.