Martin Giersberg

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens

BackgroundCoccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds.ResultsUsing the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts.ConclusionThe results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

High-level expression of a single-chain Fv fragment (scFv) antibody in transgenic pea seeds

Summary The field pea(Pisum sativumL.)is a protein-rich crop and pea seeds are well suited for the production of recombinant proteins. Here we show, that recombinant antibodies can be accumulated in transgenic pea seeds in a homozygous transgenic line up to 2 percnt; of total soluble seed protein. The expression was controlled by the seed-specific USP promoter and the transgenic single-chain Fv antibody protein was retended in the endoplasmic reticulum. The stable inheritance, shown by investigation of the high-level accumulation in the R3 offspring is another important feature of this new antibody production system. The suitability of transgenic pea seeds as an economically potent production system for recombinant antibodies is clearly demonstrated by our results.

Abscisic acid deficiency of developing pea embryos achieved by immunomodulation attenuates developmental phase transition and storage metabolism

The transition of pea embryos from pre-storage to maturation is partially controlled by abscisic acid (ABA). Immunomodulation in pea embryos specifically reduces free ABA levels during transition stages. Such seeds are, therefore, suitable models for studying ABA deficiency by global transcript and metabolite analysis. Compared with the wild type, anti-ABA seeds are smaller, contain fewer globulins and show lower dry matter accumulation and delayed differentiation. Free sugars are decreased, indicating lower uptake and/or elevated mobilisation. Lower levels of trans-zeatins suggest that ABA reduction influences rates of cytokinin synthesis and/or its level of accumulation. Abscisic acid deficiency leads to a general downregulation of gene expression related to transcription and translation. At the transcriptional level, anti-ABA embryos reveal a wide-range repression of carbohydrate oxidation, downregulated sucrose mobilisation, glycolysis and the tricarboxylic acid cycle/Krebs cycle (TCA cycle). Genes related to starch, amino acid and storage protein biosynthesis are downregulated, indicating a general decrease in metabolic fluxes. We conclude that during embryo differentiation ABA triggers broad upregulation of gene activity and genetic reprogramming, involving regulated protein degradation via the ubiquitin/proteasome system. Abscisic acid deficiency affects gene expression associated with transport processes and stimulation of membrane energisation. Our study identified mediators and downstream signalling elements of ABA during embryo differentiation, such as the transcription factor FUSCA3, SnRK1 kinase and Ca(2+) signalling processes. This suggests that ABA interacts with SnRK1 complexes, thus connecting SnRK1, sugar and stress signalling with ABA. Certain protein kinases/phosphatases known to negatively respond to ABA are upregulated in the modulated line, whilst those which respond positively are downregulated, pointing to a highly coordinated response of the gene network to ABA levels.

Covalent dimerization of camelidae anti-human TNF-alpha single domain antibodies by the constant kappa light chain domain improves neutralizing activity.

The tumor necrosis factor-alpha (TNFalpha) plays an important role in a number of chronic inflammatory disorders. Monoclonal camelidae variable heavy chain domain-only antibodies (V(H)H) have been developed to antagonize the action of human TNFalpha (anti-TNF-V(H)H). Here we describe a strategy to obtain functional dimeric anti-TNF-V(H)H molecules, based on the C-terminal fusion of a kappa light chain domain to the anti-TNF-V(H)H. The resulting fusion protein was transiently expressed by use of viral vectors in Nicotiana benthamiana((Nb)) leaves and purified. Competitive ELISA and cell cytotoxicity assays revealed that the dimerized anti-(Nb)TNF-V(H)H(Ckappa) proteins blocked TNFalpha-activity more effectively than either the monomeric Escherichia coli((Ec)) produced anti-(Ec)TNF-V(H)H or the monomeric anti-(Nb)TNF-V(H)H(Ckappa). We suggest that enhanced inhibition shown by dimeric anti-(Nb)TNF-V(H)H(Ckappa) proteins is achieved by an increase in avidity.

Production of Recombinant Antibodies in Pea Seeds and Their Oral Application in Piglets

Transgenic plants offer an effective and economically interesting alternative for the heterologous production of biomolecules for medical and veterinary purposes. Plants are free of human and animal pathogens and offer virtually unlimited scale-up potential with low initial investment. Especially the large seeds of cereal and legume crop plants represent attractive bioreactors (Giersberg et al., 2004).