Submitted to: Journal of Federation of American Societies for Experimental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/21/2006
Publication Date: 3/20/2006
Citation: Nemchinov, L.G., Paape, M.J., Sohn, E.J., Bannerman, D.D., Zarlenga, D.S., Hammond, R. 2006. Bovine cd14 receptor produced in plants reduces severity of intramammary bacterial infection. Interpretive Summary: Coliform mastitis is estimated to cause $800 million in economic losses to the dairy industry, and in the absence of effective vaccines, present control relies heavily upon antibiotics and topical germicidal chemicals, which remain sub-optimal. Therefore, there is an urgent need to develop new therapeutic reagents. A protein has been identified from milk that, when injected into the udder, is able to reduce the inflammation caused by bacterial infection. We inserted the gene for this protein into a plant virus which then produced the protein in virus-infected plants. When the purified plant-produced protein was tested in a cell assay and by injection into udders, it functioned in the same manner as the naturally occurring milk protein. Our results suggest that the plant-derived protein can be an effective therapeutic reagent for treatment of mastitis. This report will be of interest an international audience of researchers, clinicians, and representatives of industry, academia, and government organizations with an interest in plant-based systems for production of recombinant medicines and veterinary health.
Technical Abstract: The CD14 antigen is a high affinity receptor for the complex of bacterial lipopolysaccharide (LPS) and LPS-binding protein in animals. Binding of the soluble form of CD14 (sCD14) to LPS, found in the outer membrane of Escherichia coli and other Gram-negative bacteria, enhances host innate immune responses and reduces the severity of mastitis. sCD14 also facilitates clearance and neutralization of LPS, thus, protecting against an excessive immune response to LPS and the development of endotoxic shock. We expressed the sCD14 protein in plants with the aim of developing a low cost, high yield system to produce a potential animal therapeutic for mastitis treatment. A truncated form of sCD14, carrying a histidine residue affinity tag for ease of purification, was incorporated into Potato virus X for transient expression in Nicotiana benthamiana. Western Blots probed with CD14-specific antibodies demonstrated that crude plant extracts and affinity-purified samples contained immunoreactive recombinant protein of predicted molecular mass. Biological activity of the plant-derived recombinant bovine sCD14 (PrbosCD14) was demonstrated in vitro by LPS-induced apoptosis and interleukin-8 production in bovine endothelial cells, and in vivo by enhancement of LPS-induced neutrophil recruitment. Finally, in E. coli infected glands subsequently infused with PrbosCD14 or saline, lower numbers of viable bacteria were recovered from PrbosCD14 infused glands and there was an absence of clinical symptoms, thus, demonstrating therapeutic efficacy in the setting of coliform mastitis. This is the first report of a functionally active animal receptor protein made in plants and the use of a plant-derived protein as an animal therapeutic for bacterial infections.