A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein.

Poor immunogenicity of small proteins is a major hurdle in developing vaccines or producing antibodies for biopharmaceutical usage. Here, we systematically analyzed the effects of 10 solubility controlling peptide tags (SCP-tags) on the immunogenicity of a non-immunogenic model protein, bovine pancreatic trypsin inhibitor (BPTI-19A; 6 kDa). CD, fluorescence, DLS, SLS, and AUC measurements indicated that the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. ELISA results indicated that the 5-proline (C5P) and 5-arginine (C5R) tags unexpectedly increased the IgG level of BPTI-19A by 240- and 73-fold, respectively, suggesting that non-oligomerizing SCP-tags may provide a novel method for increasing the immunogenicity of a protein in a highly specific manner.

Nanometer-Sized Aggregates Generated Using Short Solubility Controlling Peptide Tags Do Increase the In Vivo Immunogenicity of a Nonimmunogenic Protein.

Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2-10 µm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (Rh) of 3-4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice's sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.

A novel coagulation inhibitor from Schistosoma japonicum.

Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µ m, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca(++). We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders.

Adverse effects of fibrin sealants in thoracic surgery: the safety of a new fibrin sealant: multicentre, randomized, controlled, clinical trial.

OBJECTIVES: The safety of fibrin sealants (FS) has been questioned in the light of recent reports of adverse effects. We evaluated the safety of a new FS in a randomized controlled trial (RCT). METHODS: Multicentre, open-label Phase II/III RCT to evaluate the safety of the new FS. The trial was approved by the Ethic Committee of each three participating Centre. FS includes two components (component 1: fibrinogen; component 2: thrombin), each of them subjected to two viral inactivation procedures. Out of 200 screened patients, 185 eligible patients (49 females, 136 males), aged between 18 and 75 years, undergoing major thoracic surgery were randomized to receive FS (#91 patients) as an adjuvant for air leak control or no treatment (#94 patients, control group). Safety variables were: percentage of subjects with adverse events associated with the therapy; formation of antibodies against bovine aprotinin; vital signs (blood pressure, body temperature, heart and respiratory rate); laboratory parameters. RESULTS: Overall operative mortality was 3.2% (6/185), 1.1% in the FS group and 5.3% in the control group, respectively. Twenty patients (22%) had adverse events in the FS group and 22 (23.4%) in the control group. Atrial fibrillation (five patients in the FS group and four in the control group) and hyperpyrexia (five and seven patients, respectively, in the two groups) were the most common adverse events. No patient reported thromboembolic events (pulmonary embolism or deep vein thrombosis) during the in hospital stay or within 1 month from discharge. None of the adverse events was considered as treatment related. The formation of bovine aprotinin antibodies was reported in a total of 34 patients (37.4%) in the FS group and was not related to any adverse effect. CONCLUSIONS: The present RCT did not show any increased risk of adverse events, and of surgical complications, related to the use of the new FS.

Postoperative complications following the Fontan procedure: the role of aprotinin.

OBJECTIVE: To determine how the anti-inflammatory properties of aprotinin impact on postoperative complications in children undergoing the Fontan procedure. METHODS: We included all patients between 14 months and 18 years (n=56) undergoing a Fontan operation at our institution between January 2005 and June 2009. The study group (n=29) included patients from January 2005 through December 2007 all of whom received aprotinin. The control group (n=27) included all patients from January 2008 through June 2009 who did not receive aprotinin. We reviewed all medical records and collected preoperative, intraoperative and postoperative data. Duration and volume of chest tube drainage were the primary outcome measures. RESULTS: Of the 20% of patients who had postoperative arrhythmias, multivariate logistic regression analysis demonstrated only aprotinin was associated with significantly decreased postoperative arrhythmias (P=0.01). Renal function and fenestration or Fontan thrombosis did not differ significantly; there was no statistically significant difference in volume or duration of chest tube drainage. Median duration of chest tube drainage was 7 days in the aprotinin group and 8 days for patients who did not receive aprotinin (P=0.36). CONCLUSION: The anti-inflammatory properties of aprotinin may be protective against postoperative arrhythmias. Aprotinin does not confer increased risks of prolonged chest tube drainage, renal dysfunction or thrombosis in patients undergoing the Fontan procedure.

Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems.

Plants have been proposed as an attractive alternative for pharmaceutical protein production to current mammalian or microbial cell-based systems. Eukaryotic protein processing coupled with reduced production costs and low risk for mammalian pathogen contamination and other impurities have led many to predict that agricultural systems may offer the next wave for pharmaceutical product production. However, for this to become a reality, the quality of products produced at a relevant scale must equal or exceed the predetermined release criteria of identity, purity, potency and safety as required by pharmaceutical regulatory agencies. In this article, the ability of transient plant virus expression systems to produce a wide range of products at high purity and activity is reviewed. The production of different recombinant proteins is described along with comparisons with established standards, including high purity, specific activity and promising preclinical outcomes. Adaptation of transient plant virus systems to large-scale manufacturing formats required development of virus particle and Agrobacterium inoculation methods. One transient plant system case study illustrates the properties of greenhouse and field-produced recombinant aprotinin compared with an US Food and Drug Administration-approved pharmaceutical product and found them to be highly comparable in all properties evaluated. A second transient plant system case study demonstrates a fully functional monoclonal antibody conforming to release specifications. In conclusion, the production capacity of large quantities of recombinant protein offered by transient plant expression systems, coupled with robust downstream purification approaches, offers a promising solution to recombinant protein production that compares favourably to cell-based systems in scale, cost and quality.

Aprotinin and anaphylaxis: analysis of 12,403 exposures to aprotinin in cardiac surgery.

BACKGROUND: Hypersensitivity reactions to the nonspecific proteinase inhibitor aprotinin may occur. The present study evaluates the incidence of hypersensitivity reactions to aprotinin. METHODS: Data were prospectively collected as part of the institution's quality assurance program. The database was screened for anaphylactic reactions, especially those against aprotinin. The definition of an allergic reaction was predefined. A severe reaction was defined as hemodynamic instability of more than 10 minutes despite high dosages of vasopressors and inotropic medication. RESULTS: Of 13,315 cardiac operations, 12,403 were done with aprotinin, with 801 reexposures in 697 patients. Eleven reactions to aprotinin (11 of 11,602; 0.09%, 95% confidence interval: 0.05% to 0.16%) were recorded after primary exposure, of which none was severe, while 12 reactions (12 of 801; 1.5%; 95% confidence interval: 0.86% to 2.6%) occurred after reexposure, of which 5 were severe. All severe reactions were in patients reexposed to aprotinin within 6 months after previous exposure. There was no reaction observed in patients reexposed to aprotinin within 3 days after the last exposure (n = 42). The incidence of hypersensitivity reactions was 4.1%, 1.9%, and 0.4% in the less than 6 months, 6 to 12 months, and more than 12 months reexposure intervals, respectively. CONCLUSIONS: The risk of hypersensitivity reactions is low after primary exposure to aprotinin. This risk after reexposure reaches a maximum between the fourth day and the 30th day after previous exposure and declines considerably after 6 months. Consequently, application of aprotinin carries a high risk between the fourth and the 30th day after previous exposure, and cannot be recommended for the first 6 months, but is justifiable in previously aprotinin-exposed patients with a high risk of bleeding after this interval.

Increased KPI containing amyloid precursor protein in experimental autoimmune encephalomyelitis brains.

Amyloid precursor protein can be translated from three alternatively spliced mRNAs. We measured levels of amyloid precursor protein isoforms containing the Kunitz protease inhibitor domain (KPIAPP), and amyloid precursor protein without the Kunitz protease inhibitor domain (KPIAPP) in brain homogenates of acute experimental autoimmune encephalomyelitis mice. At the preclinical phase of the disease, both KPIAPP and KPIAPP levels were significantly higher in homogenates from brains of autoimmune encephalomyelitis mice, whereas at the acute phase of the disease only KPIAPP remained significantly elevated compared with controls. At the recovery phase, no differences were observed between the groups. The early and isoform-specific elevation of KPIAPP in autoimmune encephalomyelitis mice suggests a possible role for amyloid precursor protein in the immune response mediating the disease.

[Anaphylactic shock related to aprotinin induced by anti-aprotinin immunoglobulin G antibody alone; report of a case].

Anaphylactic shock related to aprotinin has been reported to be induced exclusively in the presence of IgE antibody. And the possibility of anaphylactic shock induced by anti-aprotinin IgG antibody alone was controversial. In this paper, we describe the first case of anaphylactic shock induced by aprotinin-specific IgG antibody alone. A 55-year-old man underwent surgical repair of the descending aorta with the use of aprotinin at 2 months after first aprotinin usage. Immediately after initiation of cardiopulmonary bypass with the continuous infusion of aprotinin, clinical symptoms of anaphylactic reaction were found. Postoperative drug lymphocyte stimulation test for aprotinin and aprotinin-specific IgE antibody were negative, but aprotinin-specific IgG antibody was 163 mg/l and positive.

Systemic allergic reactions to aprotinin injection around the Achilles tendon.

Local aprotinin injections are used in the treatment of chronic tendinopathy. Severe allergic reactions, although uncommon, have been reported. We highlight two instances of systemic allergic reaction, and discuss the potential side effects of local aprotinin injections in the orthopaedic setting as well as the evidence base for its use.

Expression, purification, biochemical and pharmacological characterization of a recombinant aprotinin variant.

Aprotinin (GAS 9087-70-1) is known as a potent inhibitor of serine proteases such as trypsin, plasmin, tissue and plasma kallikrein. In this study, an aprotinin variant was designed by means of rationale mutagenesis that differs from aprotinin by two amino acids in the active site and by seven amino acids in the backbone. The recombinant protein is expressed in a secretory yeast system enabling large scale production. A purification procedure was developed to yield high amounts of pure and correctly processed aprotinin variant. The changes in the active site of the aprotinin variant increase the potency towards inhibition of plasma kallikrein whereas the inhibition of plasmin is only marginally reduced. The net charge of the molecule is reduced from the basic (IP 10.5) to the neutral range (IP 5.6). The recombinant aprotinin variant shows a decrease of immunogenicity in several models. No cross-reactivity with human and rabbit antibodies directed against aprotinin was observed both in in vivo and in ex vivo studies. In addition, the variant is more potent in a rat brain edema model of acute subdural hematoma compared to aprotinin.

Aprotinin and hemostasis monitoring concerns during cardiac surgery.

Aprotinin (Trasylol) is a serine protease inhibitor, isolated from bovine lung that initially was marketed for the treatment of pancreatitis. In the mid 1980s, reports of its ability to decrease hemorrhaging after cardiopulmonary bypass surgery introduced the drug to the realm of cardiac surgery. Unfortunately, its introduction into this arena was followed by the publication of multiple studies and case reports that blamed aprotinin for poor outcomes in the form of early graft closure. More than 17 years have passed since the initial article describing the use of aprotinin during cardiopulmonary bypass, and with time there has been a significant increase in scientific knowledge and clinical experience. Interestingly, modern literature does not support the dogma that aprotinin is a procoagulant. Aprotinin increases the activated partial thromboplastin time (aPTT), as well as the kaolin- and celite-activated clotting time (ACT), regardless of heparin. Aprotinin, because of its ability to inhibit kallikrein, has been found to decrease thrombin antithrombin III complexes, fibrin-split products, fibrinopeptide 1+2, prothrombin fragments, and all markers of thrombin formation. Some authors have suggested that it may have a synergistic effect with heparin to ensure graft patency. Anticoagulation monitoring during the use of aprotinin also has been developed based on early studies. Aprotinin administration does influence the results of various ACT tests, and consequently different methods of testing anticoagulation have been developed. Researchers have demonstrated that the celite ACT is not "artificially" prolonged in the presence of heparin and aprotinin, rather the kaolin ACT is "artificially" shortened. This article will review the scientific literature with regard to aprotinin's anticoagulatory effects and review the current recommendations for hemostasis monitoring during the use of aprotinin.

Bacterial lipopolysaccharide-stimulated release of tumor necrosis factor-alpha from the isolated rat heart: the effect of aprotinin and forskolin.

Aprotinin has been reported to reduce plasma levels of inflammatory cytokines associated with cardiopulmonary bypass (CPB). Because CPB is also associated with elevated levels of bacterial lipopolysaccharide (LPS) and LPS stimulates release of inflammatory cytokines from the heart we tested the hypothesis that aprotinin would inhibit cardiac release of tumor necrosis factor-alpha (TNF) provoked by LPS. Isolated rat hearts were perfused Langendorf style. After 30 minutes of equilibration LPS (100 ng/mL) was infused for 60 minutes. Timed samples of coronary effluent were collected at 0, 30, 60, 90, 120, and 150 minutes after the initiation of LPS for the measurement of coronary flow and the determination of TNF and cyclic AMP. Other hearts were perfused with buffer containing aprotinin [137 kallikrein-inhibiting units (KIU)/mL or 250 KIU/mL] and then infused with LPS. An additional group received forskolin (10 microM) and LPS. In hearts perfused as controls with buffer alone no TNF was detected in the coronary effluent. In hearts perfused with LPS TNF was reliably detected in the coronary effluent at 60 minutes (606 +/- 450 pg/min) and increased with time to a level of 1792 +/- 650 pg/min at 150 minutes. The addition of aprotinin had no significant effect on LPS-stimulated TNF release. For instance in hearts perfused with 137 KIU/mL aprotinin LPS-stimulated release at 150 minutes was 2141 +/- 732 pg/min and in hearts perfused with 250 KIU/mL LPS-stimulated TNF release was 2049 +/- 789 pg/min. Forskolin administration was associated with release of cyclic AMP from the heart and completely inhibited LPS-stimulated TNF release. We conclude that LPS stimulated release of TNF from the heart. Adding aprotinin to the perfusion buffer in either high or low concentrations did not attenuate LPS-stimulated cytokine release. Elevating myocardial cyclic AMP with forskolin completely attenuated LPS-stimulated TNF release.

Safety of aprotinin use and re-use in pediatric cardiothoracic surgery.

BACKGROUND: Hypersensitivity reactions to aprotinin have been reported in adult cardiac surgical patients undergoing initial and re-exposure to the medication. This study describes the incidence and impact of aprotinin hypersensitivity reactions in children undergoing cardiothoracic surgery. METHODS AND RESULTS: In this retrospective review of our entire experience with aprotinin (n=865), 681 first exposures, 150 second exposures, and 34 third or higher exposures were examined. Reactions were classified as mild (generalized cutaneous erythema, Type A) or severe (unexplained cardiopulmonary instability after aprotinin exposure, Type B). Records of patients sustaining a reaction were reviewed to assess the impact of the reaction on outcome and to survey reaction management strategies. Reactions occurred in 7 of 681 first exposures (1.0%), of which 2 were Type A and 5 were Type B. In second exposures, there were reactions in 2 of 150 (1.3%), of which both were Type B. In 34 third or higher exposures, there was only 1 reaction (2.9%), which was Type B. Reactions were no more likely on second, third, or higher exposure than on initial exposure. Skin testing had a negative predictive value of 98.9% and a positive predictive value of 20%. Anti-aprotinin IgE was undetectable in 7 of 8 reactor cases tested. No adverse sequelae were attributed to aprotinin reaction. CONCLUSIONS: The risk of hypersensitivity reactions to aprotinin is low in children undergoing cardiothoracic surgery, even with multiple exposures to the medication. Reactions are more likely with re-exposure, and risk increases with multiple exposures. Neither skin testing nor assays for IgE identified reactors.

Characterisation of Ag1, the major species-specific contaminant of bovine crude heparin, and its identification as an aprotinin/heparin complex.

Heparin is a potent anticoagulant polysaccharide purified for decades from ruminants or porcine tissues. However, with the emergence of bovine spongiform encephalopathy (BSE), the source of pharmaceutical heparin is currently restricted to porcine intestinal mucosa. A major species-specific contaminant, called Ag1, has recently been identified in bovine crude heparin [Rivera et al., J. Pharm. Biomed. Anal., submitted] and used to develop an enzyme-linked immunosorbent assay (ELISA) for the species origin control of crude heparins [Levieux et al., J. Immunoassay, submitted]. In this report, we describe the different investigations, which were carried out to identify Ag1. This antigen was first localised by immunohistological studies essentially in the connective tissue of the bovine small intestine. After extraction from an intestinal extract by immuno-affinity chromatography, Ag1 was isolated as a single band by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Ag1 was then partly sequenced and identified as an aprotinin/heparin complex. Aprotinin, also known as the bovine pancreatic trypsin inhibitor (BPTI), is present with heparin in mast cells, and is very resistant to heat, pH, chemical treatments and proteolytic digestion. The stability of Ag1 towards the different treatments performed during heparin extraction process allows this protein to remain in sufficient amounts in crude heparin and makes it an ideal target for the immunochemical control of the absence of bovine material in crude heparins.

Anaphylactic reactions to aprotinin reexposure in cardiac surgery: relation to antiaprotinin immunoglobulin G and E antibodies.

BACKGROUND: Aprotinin, a serine proteinase inhibitor, reduces bleeding during cardiac surgery. As aprotinin is derived from bovine lung, it has antigenic properties. This investigation examined the incidence of anaphylactic reactions in patients reexposed to aprotinin and the relation to preformed antiaprotinin immunoglobulin (Ig)G and IgE antibodies. METHODS: This prospective observational study conducted at five centers in Germany evaluated patients undergoing repeat cardiac surgery reexposed to aprotinin between 1995 and 1996. Antiaprotinin IgG and IgE antibody measurements, using a noncommercial enzyme-linked immunosorbent assay and an immunofluorescence assay, respectively, were performed preoperatively and postoperatively. An anaphylactic reaction was defined as major changes from baseline within 10 min of aprotinin administration of systolic pressure 20% or greater, heart rate 20% or greater, inspiratory pressure greater than 5 cm H2O, or a skin reaction. RESULTS: In 121 cases (71 adults, 46 children), a mean aprotinin reexposure interval of 1,654 days (range, 16-7,136 days) was observed. Preoperative antiaprotinin IgG (optical density ratio > 3) and IgE antibodies (radioallergosorbent test [RAST] score < 3) were detected in 18 and 9 patients, respectively. High concentrations of each (IgG, optical density ratio > 10; IgE, RAST score > or = 3) were detected in five patients. Three patients (2.5%; 95% confidence interval, 0.51-7.1%) experienced an anaphylactic reaction after aprotinin exposure, followed by full recovery; these patients had reexposure intervals less than 6 months (22, 25, and 25 days) and the highest preoperative IgG concentrations of all patients (P < 0.05). Assay sensitivity was 100%, as no anaphylactic reactions occurred in IgG-negative patients (95% confidence interval, 0.0-3.1%); assay specificity was 98%. Preoperative IgE measurements were quantifiable in two of three reactive patients and in three nonreacting patients. CONCLUSIONS: Quantitative detection of antiaprotinin IgE and IgG lacks specificity for predictive purposes; however, quantitation of antiaprotinin IgG may identify patients at risk for developing an anaphylactic reaction to aprotinin reexposure.

Immunolocalisation of BPTI-like serine proteinase inhibitory proteins in mast cells, chondrocytes and intervertebral disc fibrochondrocytes of ovine and bovine connective tissues. An immunohistochemical and biochemical study.

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus

An immediate, allergic skin reaction to aprotinin after reexposure to fibrin sealant.

BACKGROUND: The safety of fibrin tissue adhesives has been a concern since they entered wide clinical application. Most commercially available kits contain the proteolytic inhibitor, aprotinin, to stabilize the fibrin clot. A bovine protein, this substance has an allergenic potency. CASE REPORT: This case report presents a patient who had a generalized allergic skin reaction, probably triggered by aprotinin upon reexposure to fibrin sealant injected subgaleally to achieve closure of a liquor fistula after neurosurgical treatment. The serologic investigation revealed aprotinin-specific IgE and IgG. From 1990 through 1998, reports of five allergic reactions following 1 million exposures to fibrin sealant were made to the manufacturer. The clinical relevance of allergic reactions to aprotinin contained in fibrin sealants and measures to avoid them are discussed. CONCLUSION: These hypersensitivity reactions are extremely rare (incidence, 0.5/100,000 for all reactions and 0.3/100,000 for serious reactions), but they must be kept in mind as possible adverse events after repeated applications of fibrin sealants within a few weeks.