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Bovine CD14 receptor produced in plants reduces severity of intramammary bacterial infection

Lev G. Nemchinov^*, Max J. Paape, Eun J. Sohn, Douglas D. Bannerman, Dante S. Zarlenga and Rosemarie W. Hammond^*,1

USDA, ARS, Beltsville Agricultural Research Center,
^* Molecular Plant Pathology Laboratory,

Bovine Functional Genomics Laboratory, Beltsville, Maryland, USA

¹Correspondence: USDA ARS Molecular Plant Pathology Laboratory, Rm. 214 Bldg. 004 BARC-West, Beltsville, MD 20705, USA. E-mail: hammondr{at}ba.ars.usda.gov

CD14 is a high-affinity receptor protein for the complex of bacterial LPS (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, reduces the severity of mastitis, and facilitates clearance and neutralization of LPS, thus protecting against an excessive immune response to LPS and development of endotoxic shock. A truncated form of sCD14, carrying a histidine residue affinity tag for purification, was incorporated into Potato virus X for transient expression in Nicotiana benthamiana plants. Western blots probed with CD14-specific antibodies demonstrated that crude plant extracts and affinity-purified samples contained immunoreactive sCD14. Biological activity of plant-derived recombinant bovine sCD14 (PrbosCD14) was demonstrated in vitro by LPS-induced apoptosis and interleukin (IL) -8 production in bovine endothelial cells, and in vivo by enhancement of LPS-induced neutrophil recruitment. Finally, in PrbosCD14-infused glands subsequently infected with E. coli, lower numbers of viable bacteria were recovered and there was an absence of clinical symptoms, demonstrating prophylactic efficacy of PrbosCD14. This is the first report of a functionally active animal receptor protein made in plants and the prophylactic use of a plant-derived protein to reduce the severity of bacterial infections in animals.—Nemchinov, L. G., Paape, M. J., Sohn, E. J., Bannerman, D. D., Zarlenga, D. S., Hammond, R. W. Bovine CD14 receptor produced in plants reduces severity of intramammary bacterial infection.

Key Words: potato virus X • plant-derived therapeutic • coliform mastitis • apoptosis • LPS

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