Eilleen S. Tackaberry

Improved Yields of Factor VIII from Heparinized Plasma

Abstract. The Factor VIII procoagulant activity in plasma, cryoprecipitate, and their polyethylene glycol (PEG) precipitates is markedly increased if blood is collected into heparin rather than into citrate phosphate dextrose (CPD). There is a 34% increase in the initial level of the Factor VIII activity in the heparinized plasma with 78% of this initial activity (184 U) recovered in the cryoprecipitate. As well, the stability of the Factor VIII activity is improved: after 24 h of incubation at 22°C, 99% of the initial activity is retained in heparinized plasma whereas only 64% remains in CPD plasma. The cryoprecipitate prepared from heparinized plasma is equally stable after 24 h. The PEG concentrate prepared from the cryoprecipitate of heparinized plasma is increased to 128 U compared to only 54 U from CPD plasma. Relative recoveries were 531 U/l for heparinized plasma versus 215 U/l for CPD plasma. This represents a 147% increase in yield.

Incorporation of plasticizer into red cells during storage

The development of flexible plastic blood bags has permitted effective blood component production and therapy. However, the plasticizer di(2‐ ethylhexyl)phthalate (DEHP), whose toxicity in humans is still undefined, is known to leach from the plastic into stored blood. Despite the availability of bags made of plastics not using DEHP, the collection and storage of red cells is still done in DEHP plasticized packs, and in fact the storage life for red cells has recently been increased up to 49 days using new anticoagulant‐preservative solutions. We examined the relationship between DEHP and stored red cells. We found that 28 percent of available 14C‐DEHP binds immediately to sites in both the membrane and cytosol fractions of the red cells, and that the total amount and distribution of 14C‐DEHP does not change significantly over 7 days. When red cell concentrates were stored with or without DEHP, using either plastic (polyolefin) bags not containing DEHP or glass, definite reduction in the osmotic stability of the red cells was found in the absence of DEHP. Plasma‐free hemoglobin levels were 90.3 mg per dl after 35 days of storage in plastic packs containing DEHP and 181.7 mg per dl in the polyolefin bags. The advantages of improved in vitro stability of red cells stored in plastics containing DEHP must be weighed against the potential hazards of patient exposure to DEHP.

Sorting of glycoprotein B from human cytomegalovirus to protein storage vesicles in seeds of transgenic tobacco.

As part of ongoing studies into the use of plant expression systems for making human therapeutic proteins, we have successfully expressed the major glycoprotein, gB, of human cytomegalovirus (HCMV) in transgenic tobacco plants. Viral glycoprotein was detectable in the protein extracts of mature tobacco seeds using neutralizing and non-neutralizing monoclonal antibodies specific for gB. Although several mammalian proteins have been expressed in tobacco, localization of these proteins in transgenic tobacco tissue has not been extensively examined. The objective of this study was to identify the site(s) of recombinant gB deposition in mature tobacco seeds. Using immunogold labelling and electron microscopy, we found specific labelling for gB in the endosperm of transgenic seeds, with gB localized almost exclusively in protein storage vesicles (PSV). This occurred in seeds that were freshly harvested and in seeds that had been stored for several months. These data indicate that gB behaves like a plant storage protein when expressed in tobacco seeds, and provide further support for the suitability of plants for producing recombinant proteins of potential clinical relevance.

Biologically active human GM-CSF produced in the seeds of transgenic rice plants

Rice flour is a well-known and characterized source of pharmaceutical ingredients, which are gluten-free and incorporated in many drug delivery applications such as excipient starch. To further exploit this uniqueness, the synthetic capacity of rice endosperm tissue, the basis of rice flour, was extended by genetic transformation. Recombinant human GM-CSF, a cytokine used in treating neutropenia and with other potential clinical applications, has been expressed in transgenic rice seeds using a rice glutelin promoter. Rice seeds accumulated human GM-CSF to a level of 1.3% of total soluble protein. The rice seed-produced human GM-CSF was found to be biologically active when tested using a human cell line TF-1. Use of rice as a host plant offers not only attractive features of safe production in seeds but also self-containment of foreign genes, as rice is primarily a self-pollinated crop plant.

DDAVP-induced release of von Willebrand factor from endothelial cells in vitro: the effect of plasma and blood cells.

The vasopressin analogue 1-deamino-8-D-arginine vasopressin (DDAVP) causes an immediate, transient rise in plasma levels of von Willebrand factor (vWF) after its administration. Although it is recognized that vascular endothelial cells play an essential role in this process, the molecular basis of the response is not understood. We have investigated the phenomenon using human umbilical vein endothelial cells as an in vitro model. When normal individuals were stimulated with DDAVP, plasma from blood samples collected subsequently caused the release of vWF from cultured endothelial cells over a 24 h period (22-46% increase over baseline), compared to control plasma (5-17%). DDAVP added directly to the endothelial cells produced no increase in vWF release. When whole blood was treated in vitro with DDAVP, and the plasma subsequently added to endothelial cells, a significant increase in vWF secretion was found. Peripheral blood mononuclear cells were then tested. In the presence of DDAVP, an increased response occurred. Further fractionation of these cells showed that monocytes were largely responsible, causing an increased vWF release of 162% at 2 h. These observations were reinforced by finding that the supernatants of monocytes incubated with DDAVP were also effective in causing increased vWF release (118% compared to 58% for the control sample). Our studies suggest that DDAVP plays an indirect role in causing the release of vWF from endothelial cells, and that peripheral blood monocytes may act as intermediary target cells, which then produce factor(s) acting directly on endothelial cells.

Biological activity of human granulocyte-macrophage colony stimulating factor is maintained in a fusion with seed glutelin peptide

Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants. Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression. Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5 end. With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active. Furthermore, native tobacco seed extracts inhibited the activity of E. coli-derived GM-CSF in this cytokine-dependent cell line. The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited. The feasibility of oral delivery of such stable seed-produced cytokines is discussed.

Pharming vaccines for hepatitis and cytomegalovirus: Towards the development of multivalent and subunit vaccines for oral delivery of antigens

A plant based high fidelity vaccine production system is being developed with emphasis on producing antigens capable of being orally delivered in multivalent or subunit plant packets. Plant-based edible vaccines may provide an attractive, safe and inexpensive alternative to conventional vaccine production. Edible plant tissues are not normally antigenic in nature. However, foreign antigens from common infectious organisms like hepatitis-B virus (HBV) can be produced along with naturally occurring storage proteins in DNA-transformed plants. Upon administration via the oral route, these transgenic plant tissues may mobilize the protective humoral and mucosal immune responses to challenge the natural infectious agent. When tobacco, carrot and rice plants were transformed with the truncated version of the HBV nucleocapsid gene expression construct, non-infective hepatitis B viral core particles were observed via electron microscopy. A second plant codon-optimised HBV expression construct was designed that included the extensin signal sequence for augmented HBV particle accumulation. Upon transformation of tobacco plants with the codon-optimised construct, over 4 times more transgenic plants with high levels of expression of the HBV nucleocapsid protein were generated in comparison with a similar vector containing the unmodified wild-type HBV gene codon sequence. Further analysis via Western blotting confirmed the presence of the viral antigen in the total protein extracts from transgenic tobacco leaves and seeds. Electron microscopy showed that the expressed protein self-assembled into viral-like particles of 25–30xa0nm in diameter. To develop an edible subunit vaccine in plant seeds, a third plant transformation construct was used for the synthesis of the human cytomegalovirus glycoprotein B (HCMV gB) subunit. The gB protein derived from tobacco seeds retained critical structural features including epitopes for neutralizing antibodies and was targeted to the protein storage vesicles of tobacco seed endosperm. Two different strains of mice were orally immunized with tobacco seeds containing low concentrations of HCMV gB, with varying dosages, but without adjuvant. No anti-gB response was detected in intestinal or serum samples. However, a systemic immune response to normal tobacco seed proteins was observed in both strains of mice. While higher expression levels of antigens in seeds must be achieved, seeds may provide an effective and immunostimulatory vehicle for delivering edible vaccines to the intestinal mucosa. One of the outstanding challenges includes defining optimum conditions of antigen presentation, dosage and immunization schedules that will induce strong mucosal and/or systemic immune responses in heterogeneous populations. Here we review the different strategies being employed to produce specific oral antigens in plant tissues.

Sustained expression of human cytomegalovirus glycoprotein B (UL55) in the seeds of homozygous rice plants.

Production of recombinant subunit vaccines in transgenic plants may be a means of reducing vaccine costs while increasing availability and safety. A plant-derived product found safe and effective for oral administration would provide additional advantages when used as a vaccine. Outstanding issues with the technology include transgene stability through successive generations and consistent bioproduction. We previously reported expression of glycoprotein B (gB) of human cytomegalovirus in seeds of transgenic tobacco. Here the goal was to determine if gB could be similarly expressed in rice, and if so, to examine expression over several plant generations. Results show that immunoreactive gB was successfully expressed in transgenic rice seeds, with sustained expression over three generations. The gB contained several neutralizing epitopes and was stable over 27xa0months.

Monoclonal anti-idiotypes for the rapid detection of human cytomegalovirus

A novel enzyme-linked immunosorbent assay (ELISA) was developed for human cytomegalovirus (HCMV) utilizing a monoclonal anti-idiotype specific for CMVB1, an antibody to HCMV. Samples of HCMV were measured by their inhibition of the binding of CMVB1 to anti-idiotype. The ELISA detected HCMV in a concentration-dependent manner from 20 to 0.6 x 10(3) PFU/ml, with 50% inhibition at approx. 3 x 10(3) PFU/ml. These data demonstrate the potential of anti-idiotype antibodies as the basis of simple and rapid diagnostic tests for infectious agents.

Human monoclonal antibodies to cytomegalovirus recognize viral epitopes on the surface of virus-infected cells

Four human monoclonal antibodies directed against human cytomegalovirus were produced by fusing Sp2/HPT heteromyeloma cells with peripheral blood lymphocytes, after stimulation in vitro for 6 days. The human hybridomas have been maintained in culture for one year and secrete, when cultured in serum-free medium, between 3.1 and 8.1 micrograms/ml of antibodies/10(6) cells/24 hours. HCV-1 and HCV-2 are IgG kappa, while HCV-3 and HCV-4 are IgG3 lambda. The four monoclonal antibodies immunoprecipitate a viral protein of 64 kD. Kinetic studies using indirect immunofluorescence indicate that this antigen appears late in the viral infectious cycle. All four monoclonal antibodies recognize human cytomegalovirus prototype strains AD-169, Davis and Towne, and 14 clinical isolates collected between 1984 and 1987. No reactivity was observed with other human herpesviruses. While no neutralizing activity could be observed with the human monoclonal antibodies, binding assays on unfixed infected cells showed that they recognized viral epitopes located on the cell surface membrane. These hybridomas may be useful for future therapy of immunocompromised patients.