Human Immune Protein CD14 Expressed in Tobacco

Source from:  Wednesday, July 05, 2006 By David R. Blais & Illimar Altosaar 07/06/2006

Four out of five people turn to plants to treat themselves when sick, relying virtually exclusively on traditional herbal medicine for their primary health care (1). It is primarily in the more privileged and developed countries that synthetic chemicals are supplementing the herbalists' apothecaries. Still, a full quarter of prescription drugs are sourced directly from plant seeds, roots, leaves, stalks, and exudates. Our food biochemistry lab, established in 1978, has worked on the premise that by genetically engineering plants already equipped with useful medical compounds it may be possible to produce hard-to-get drugs. Hence, we have embarked on a program to engineer plants that promote protection against pathogens. CD14 protecting mucosal surfaces Plants and animals are constantly exposed to a multitude of microorganisms that colonize their surfaces. In general, a symbiotic relationship develops between the host and the microbe that provides benefits and advantages to each organism. However, pathogenic microorganisms often extend their colonizing capacities beyond mucosal surfaces. These pathogenic invasions are initially countered by innate defense mechanisms that preexist in the host and act within minutes after the initial infection. The innate immune response is based on an early recognition of microbe-specific motifs, known as pathogen-associated molecular patterns (PAMP). Most PAMPs are highly conserved surface-derived molecules, such as lipopolysaccharide or LPS, a major component of the outer membrane of Gram-negative bacteria. In mammals, this early detection is mediated by pattern recognition receptors, such as CD14 (cluster of differentiation 14), capable of detecting picomolar concentrations of LPS. By binding to LPS, CD14 and the Toll-like receptor 4 (TLR4) complex promote inflammatory responses at the site of infection through the secretion of pro-inflammatory mediators and the recruitment of immune cells. The ubiquitous presence of CD14 at mucosal surfaces and secretions (i.e., tears, cornea, breast milk, saliva, lungs, intestine, urine, sperm, and amniotic fluid) reflects its importance to the host in fighting Gram-negative infections in these constantly challenged mucosal environments (2,3). Given its widespread mucosal distribution, numerous medical applications have been suggested for CD14, such as reducing the severity of LPS-induced septic shock and preventing Gram-negative infections at mucosal surfaces (4). Despite several attempts in different expression systems, recombinant human CD14 is still not mass produced for clinical evaluation due to obstacles concerning expression levels, production cost, as well as protein stability and activity (reviewed in reference 5). Plant-produced rhCD14 could prevent ocular infections By targeting the expression of the human CD14 coding sequence in the endosperm of tobacco seeds under the control of glutelin promoters, we were able to obtain significant levels (16 µg rhCD14/g of seeds) of recombinant human CD14 (rhCD14) proteins (5). Plant-produced rhCD14 was efficiently stored in a stable and biologically active form in tobacco seeds. When exposed to LPS from Pseudomonas aeruginosa, an ocular Gram-negative pathogen, plant-produced rhCD14 was able to induce an innate immune response of corneal epithelial cells similarly to CD14 naturally found in human tears (3,5). These findings offer some promising applications for plant-made rhCD14 in preventing bacterial keratitis among contact lens users, and for use during ocular interventions, such as laser eye surgery or suppressing implant infections after orbital surgery (6). Such applications could include the addition of rhCD14 to contact lens solutions or artificial teardrops to mimic the immune advantages of human tears in reducing the risk of developing ocular Gram-negative infections. Plant-made rhCD14 for the fortification of infant milk formulas Because the tobacco endosperm glycosylation of rhCD14 might affect its stability and half-life, digestibility experiments were performed with proteolytic enzymes to determine if plant-made rhCD14 had an increased susceptibility to digestion. In vitro pepsin and pancreatin digestion, to mimic the human newborn gastrointestinal tract, revealed that plant rhCD14 has a proteolytic resistance similar to that of CD14 present in human breast milk (2,5). The slight variation between plant rhCD14 and breast milk CD14 proteins might be attributed to the environmental milieu of the protein, such as the presence of protease inhibitors in breast milk. With its similar digestive susceptibility, rhCD14 produced in a food crop (or in a non-food plant for confinement issues) could one day be added to commercial infant milk formulas to mimic the immune advantage of breast milk for the neonate and to creams for cracked nipples to prevent mammary infections during lactation. This latest potential application of 'humanizing' infant milk formula with plant-produced breast milk proteins, such as rhCD14, might help reduce mother-to-child transmission of HIV in resource-constrained countries.7 No safe newborn feeding methods are currently available for HIV-infected mothers in these countries since breast milk is a vehicle of HIV transmission and infant formula is a source of gastrointestinal illnesses due to the lack of breast milk protective immune factors and unsanitary conditions during formula preparation. There is therefore an urgent need to ensure that breast milk substitutes embody optimal health benefits. To do so, transgenic food crops, such as rice (Fig. 1), could cheaply and safely reconstitute the beneficial and protective breast milk proteome in infant formulas to replicate the immune protection of breast milk against gastrointestinal illnesses while keeping the HIV virus at bay (7). At least seven breast milk proteins with immune properties have already been produced in transgenic food crops (7). With our current knowledge of the breast milk proteome and its fate in the newborn gastrointestinal tract, along with the maturing plant biotechnology sector, production of dozens more immune proteins in transgenic food crops is deemed feasible to supplement such fortified formulas with pharma flour. Future perspectives Plant-made rhCD14 was efficiently stored in a stable and biologically active form in transgenic tobacco seeds. Transgenic rice, expressing the codon-optimized version of the hCD14 coding sequence, along with stronger promoters and signal sequences, such as the KDEL retention signal, are currently being developed in our lab to further increase the yields of the recombinant protein and allow direct oral delivery (Fig. 1). This expression system constitutes a promising source of rhCD14 to continue identifying the roles of this LPS receptor protein in innate immune responses at mucosal surfaces in addition to the aforementioned potential preventive and therapeutic medical applications. Enditem