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.

Development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco.

Plant seeds offer unique opportunities for the production and delivery of oral subunit vaccines. We have used the immunodominant glycoprotein B complex of human cytomegalovirus (HCMV), introduced into tobacco plants, as a model system for studying the merit of this promising approach. Given the advantages of expressing proteins in seeds, a novel expression vector was developed incorporating regulatory sequences of glutelin, the major rice seed storage protein, to direct synthesis of recombinant glycoprotein B. Analysis of genomic DNA of 28 selected tobacco transformants by PCR amplification showed that 71% harboured the gB cDNA, a finding further documented by Southern blotting. Specific immunoassays of protein extracts from seeds of positive plants showed that all were producing antigenic glycoprotein B at levels ranging from 70-146 ng/mg extracted protein. In addition, similarity with native glycoprotein B produced in HCMV-infected cells was also demonstrated by inhibition of immunofluorescence on HCMV-infected human fibroblasts. These data are the first to report the expression of an immunodominant antigen of HCMV in plant tissues, indicating the fidelity with which this very large heterologous viral glycoprotein can be synthesized in this model system.

Anti-idiotypic mimicry of a neutralizing epitope on the glycoprotein B complex of human cytomegalovirus.

Experiments were carried out to investigate the ability of rabbit anti-idiotype antibodies (Ab2), directed against an anti-human cytomegalovirus monoclonal antibody (Ab1), to induce neutralizing antibodies specific for the immunodominant glycoprotein B viral complex. Mice immunized with Ab2 produced anti-Ab2 (Ab3) that was both antigen and idiotype specific with regard to Ab1. We conclude that the Ab2 antibodies mimicked a neutralizing epitope and acted as a network antigen for inducing a specific anti-human cytomegalovirus antibody response in this experimental system.

A novel approach to the laboratory diagnosis of Chlamydia trachomatis infections using monoclonal anti-idiotypic antibodies.

We have developed a novel enzyme immunoassay (EIA) for the specific detection of Chlamydia trachomatis utilizing a monoclonal anti-idiotypic antibody to an antibody directed against a chlamydia specific epitope on 60 kDa heat-shock protein (HSP60). The basis of the assay is the inhibition of the binding of idiotype to anti-idiotype by antigen present in test samples. Two configurations of the assay were developed: a blocking EIA and a competition EIA. Greater sensitivity was observed using the competition EIA, with the assay detecting purified recombinant HSP60 and purified chlamydia in a concentration-dependent manner from 0.01 to 10 micrograms protein and from 0.5 to 12 micrograms total protein, respectively. The assay is highly specific and offers several potential advantages over currently available EIAs for the detection of this pathogen.

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.

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.

Human factor VIII from heparinized plasma. Purification and characterization of a single-chain form.

Human factor VIII was purified from heparinized blood by cryoprecipitation, poly(ethyleneglycol) precipitation, Affi-Gel blue, aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. A purification of 280,000-fold over plasma with a specific activity over 5300 units/mg was achieved. Analyses of factor VIII using HPLC indicated a molecular mass of 280-340 kDa. Variation in the native mass may reflect heterogeneity of the protein due to associated lipid since structural analysis confirmed that factor VIII contained variable amounts of free fatty acids and diglycerides and triglycerides, but no phospholipids. Additional characterization by denaturing polyacrylamide gel electrophoresis under reducing conditions, followed by silver staining, showed a major single-chain polypeptide of factor VIII with a mass of approximately 260 kDa. To determine whether proteolyzed forms of factor VIII were present during fractionation, we analysed earlier steps in purification. This revealed additional species of factor VIII eluting faster than the single-chain form during chromatography on polyelectrolyte E5. Gel electrophoresis showed that these species of factor VIII consisted of multiple polypeptide chains, and partial peptide mapping using Staphylococcus aureus V8 protease indicated that they were structurally related. Monoclonal and hemophilic antibodies were used in immunoadsorption experiments to demonstrate that the purified factor VIII was composed predominantly of the 260-kDa factor VIII chain.

Measurement of human factor VIII by avidin-biotin dot immunobinding ELISAs.

Hemophilia A is a congenital bleeding disorder which is characterized by a functional deficiency of the coagulation protein factor VIII. We have developed sensitive enzyme-linked immunosorbent assays (ELISAs) for measuring the antigenic reactivity of factor VIII. The assays utilize dot immunobinding techniques, commercial monoclonal antibodies, and a detection system enhanced by the interaction of avidin and biotin. The dot immunobinding ELISAs were optimized for measuring factor VIII in normal and hemophilic plasma, and in partially and highly purified preparations of factor VIII. Linear standard curves were established for all samples, defining the range for accurate measurement. Factor VIII was detected at concentrations as low as 0.0005 U/ml, which represents 0.1 pg of protein.

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.

Rapid controlled thawing of fresh-frozen plasma in a modified microwave oven.

A microwave oven has been specifically modified to permit rapid thawing of fresh-frozen plasma (FFP) by using a rotating disc with a temperature sensor to hold the plasma bag. This modification makes it possible to mix the FFP continuously during thawing, and automatically shuts the oven off when the plasma reaches 21 degrees C. Comparisons were made between FFP thawed in the modified microwave oven and FFP thawed conventionally in a 37 degrees C waterbath. The following tests were done: total protein, albumin, and immunoglobulin concentrations; plasma fibrinogen, factor VIII, and factor IX activities; protein electrophoresis, albumin aggregation, hemolytic complement activity, and plasma particle count and size. In no case was there a significant difference between plasma thawed in the microwave oven compared with that thawed in the waterbath. Further, microwave thawing was reliable and rapid; all units of FFP thawed in less than 6 minutes, and the thawed plasma did not vary by more than 6 degrees C from the preselected final temperature of 21 degrees C. Thus, it appears that controlled thawing of FFP in a microwave oven specifically designed for this purpose is an effective and reliable method and has many advantages over conventional thawing of FFP.

Stability of VIII:C in plasma: the dependence on protease activity and calcium.

Loss of Factor VIII procoagulant activity (VIII:C) following blood collection is a major problem in providing sufficient amounts for therapeutic use and biochemical analyses. We have examined the effects of inhibition of plasma proteases and maintenance of physiological calcium ion on plasma VIII:C stability. The addition of protease inhibitors such as benzamidine, phenylmethylsulfonyl fluoride (PMSF), aprotinin, or soybean trypsin inhibitor (SBTI) to CPD plasma provided no significant protection against decay of VIII:C activity. Neither the rate of decay in the first 24 hours nor the final VIII:C activity observed after storage for 48-72 hours were significantly altered. On the other hand, addition of DFP or heparin to CPD plasma resulted in a marked improvement in VIII:C stability over 24 hours. This demonstrated that these two inhibitors are effective in preventing VIII:C degradation during storage. In addition to protease inhibition, the importance of maintaining physiological calcium ion was demonstrated by 100% stabilization of VIII:C in heparin plasma. Plasma obtained from CPD plus heparin blood could also be stabilized provided free calcium ion levels were restored to physiological concentrations. The inactivation of VIII:C in CPD plus heparin plasma was completely reversible up to 4 hours after collection. Studies on the recovery of activity after recalcification of CPD plus heparin plasma provided kinetic data which support a renaturation process of VIII:C rather than one due to enzymatic activation. The use of a thrombin-specific chromogenic substrate revealed that after recalcification and during the recovery of VIII:C activity, there was no significant thrombin activity. Although the data suggest that proteolytic degradation plays some part in VIII:C decay, only the maintenance of physiologic calcium ion levels under cover of an effective non-chelating anticoagulant and protease inhibitor allows preservation of VIII:C activity.

Improved yields of factor VIII from heparinized plasma.

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 cryoprecipitated. As well, the stability of the Factor VIII activity is improved: after 24 h of incubation at 22 degrees 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.