Targeting ETEC-related post-weaning diarrhoea in piglets
Postweaning diarrhoea (PWD) in pigs is responsible for important economic losses in the global pig rearing industry. This illness is predominantly caused by enterotoxigenic E. coli strains carrying the F4 (ETEC-F4) and F18 (ETEC-F18) fimbriae. There is no treatment available to fight PWD except using antibiotics. However, the prophylactic use of antibiotics is forbidden in Europe and is expected to be forbidden in the rest of the world because of increasing abundance of resistance genes. Delivery of IgAs to mucosal surfaces as passive immunotherapy agent is a very promising strategy to prolong maternal lactogenic immunity against post-weaning infections as we previously described in Virdi et al. 2013. In this work, anti-ETEC-F4 antibodies were engineered from phage display-selected variable domains from Henri de Greve’s lab in the department of structural Biology in Brussels, in a simplified IgA (sIgA) format. These antibodies were then produced in seeds of Arabidopsis thaliana and scaled-up to test their potential in passive immunization assays in vivo. For this, crushed seeds were incorporated in the feed of weaned piglets and showed to be protective against ETEC-F4 challenge. Following the same rationale, antibodies against ETEC-F18 are currently being developed.
In addition, we plan to evaluate the stability of the antibodies during feed processing, mimicking the conditions to which seeds are subjected in the common feed processing chain. Also, the functionality of the orally fed antibodies in fecal matter will be studied. Evaluation of the presence and functionality of the plant made antibodies in the fecal samples will allow us to determine the stability during gut transit.
Elite antibodies against both F4 and F18 ETEC strains are currently being transformed and/or scaled up in soya for further valorization (see section 1).
Development of maternal and piglet anti-ETEC immunity
In most commercial farms sows are vaccinated against enterotoxigenic Escherichia coli (ETEC), including F4-ETEC. This immunization is essential to stimulate the production of specific antibodies and their transfer to the piglets via colostrum and milk to prevent diarrhea during the neonate period and shortly after weaning. By determining and quantifying the different isotypes of ETEC specific antibodies in sow’s blood serum and colostrum/milk and the blood serum and stool of their suckling piglets (before and after weaning), we aim to obtain a better basic understanding of the transfer and role of the maternal protective antibodies. Moreover it will help us evaluate the antibody dosage requirement for the recombinant antibody-feed formulation.
Passive immunization against the human respiratory syncytial virus
Delivery of antibodies at the mucosal surfaces as passive immunotherapeutic agents is a promising strategy to prevent and treat infectious diseases. Human Respiratory Syncytial Virus (HRSV) is one of the major causes of lower respiratory tract infections in infants. HRSV can cause severe disease and even death in frail individuals such as very young children and the elderly. Prophylactic treatment with an HRSV targeting monoclonal antibody is clinically approved. However, the high cost that is associated with the current mammalian cell-based manufacturing systems hampers the widespread implementation of this therapy. Alternative expression platforms such as transient expression in plants not only provide higher scalability and reduced costs but also permit a rapid evaluation of different antibody versions. In this work we will evaluate the efficacy of twelve different anti-HRSV antibody variants produced in Nicotiana benthamiana leaves.
Antibodies against HRSV have been built by the combination of three different Nanobodies® (VHH) (Schepens et al., 2011) genetically fused to the Fc fragment of different murine and human monomeric IgA and IgG antibodies. Furthermore, IgA based antibodies will also be expressed and tested in their secretory form (SIgA).
Engineering of these different antibody versions has been greatly facilitated by using the GoldenBraid2.0 (GB2.0) technology (Sarrion-Perdigones et al., 2013).
Sarrion-Perdigones A. et al. (2013). GoldenBraid2.0: A comprehensive DNA assembly framework for Plant Synthetic Biology. Plant Physiol. 162, 1618-1631.
Schepens B. et al. (2011). Nanobodies® Specific for Respiratory Syncytial Virus Fusion Protein Protect Against Infection by Inhibition of Fusion. J Infect Dis. 204, 1692-1701.
Virdi V. and Depicker A. (2013). Role of plant expression systems in antibody production for passive immunization. Int. J. Dev. Biol. 57, 587-593.
Virdi V. et al. (2013). Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection. Proc. Natl. Acad. Sci. USA 110, 11809-11814.