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ABSTRACT: A highly purified, liquid, 10% immunoglobulin product stabilized with proline, referred to as IgPro10 has recently been developed. IgG was purified from human plasma by cold ethanol fractionation, octanoic acid precipitation and anion-exchange chromatography. The manufacturing process includes two distinctly different partitioning steps and virus filtration, which were also assessed for the removal of prions. Prion removal studies used different spike preparations (brain homogenate, microsomes, purified PrP(sc)) and three different detection methods (bioassay, Western blot, conformation-dependent immunoassay). All of the investigated production steps were shown to reduce significantly all different spike preparations, resulting in an overall reduction of >10log(10). Moreover, the biochemical assays proved equally effective to the bioassay for the demonstration of prion elimination. Four of the manufacturing steps cover three different mechanisms of virus clearance. These are: i) virus inactivation; ii) virus filtration; and iii) partitioning. These mechanisms were assessed for their virus reduction capacity. Virus validation studies demonstrated overall reduction factors of >18log(10) for enveloped and >7log(10) for non-enveloped model viruses. In conclusion, the IgPro10 manufacturing process has a very high reduction potential for prions and for a wide variety of viruses resulting in a state-of-the-art product concerning safety towards known and emerging pathogens.
Biologicals 07/2008; 36(4):239-47. · 1.70 Impact Factor
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ABSTRACT: Nanofiltration has proven to be an effective technology for virus removal. The small nonenveloped viruses, such as HAV and parvovirus B19 (B19), are evidently more difficult to remove than the larger enveloped viruses. However, since antibodies against these viruses, HAV and B19, are constantly present in large plasma pools, it is conceivable that antibody binding will lead to an increase in size of the viruses due to a corona of antibodies on the virus surfaces. These enlarged viruses should be easier to remove by nanofiltration than naked viruses. In this study, antibody-coated viruses and free virions were subjected to nanofiltration to determine whether the antibody-mediated increase in size contributes to the elimination of viruses which are otherwise too small to be retained by filters of a given nominal pore diameter.
One-percent IgG or 1-percent albumin solutions were spiked with bovine enterovirus (BEV) and filtered through 50-nm filters. This virus cross-reacts with antibodies in pooled human IgG solutions but persists as free virions in albumin solutions. Similarly, two parvoviruses, bovine parvovirus (BPV) and minute virus of mice (MVM), in 1-percent IgG solutions were filtered through 20-nm filters. BPV cross-reacts with antibodies in human IgG, whereas MVM remains unaffected. Virus removal was assessed by in vitro infectivity assays. Two methods were developed to displace the antibodies from BEV and BPV and thereby render them accessible for titration.
Antibody-coated BEV was eliminated to below detection limit by the 50-nm filters (logarithmic reduction factors [LRF] > or = 5), while free BEV virions passed without restraint through the same filters. Similarly, antibody-coated BPV was completely retained by the 20-nm filters (LRF > or = 5). MVM virions were strongly retained by the 20-nm filters; however, the virus was still detectable in the filtrate.
These results indicate that viruses with bound antibodies are efficiently eliminated by nanofiltration by use of filters having nominal pore sizes larger than the diameter of the respective free virions.
Transfusion 08/2002; 42(8):1005-10. · 3.22 Impact Factor
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ABSTRACT: Virus inactivation by ethyleneimines was first introduced more than 30 years ago. Selective targeting of nucleic acids was reported for oligomeric ethyleneimines. In this study, trimeric ethyleneimine (TEI) was used to inactivate minute virus of mice (MVM; Parvoviridae) and Semliki forest virus (SFV; Togaviridae). The pH-dependency of the inactivation kinetics observed with MVM was different compared to the kinetics reported for other viruses. The higher inactivation rate at higher pH favoured the idea of a mechanism involving protein modifications. Alteration of the isoelectric point and changes in mass could be observed after treatment of soluble proteins with TEI. The uptake of MVM by host cells was reduced or completely blocked by TEI treatment, as shown by monitoring viral internalisation of DNA into target cells. The observed loss of virus infectivity coincided with the inhibition of virus uptake. Thus, virus inactivation by TEI is most likely also a result of chemical modifications of viral surface proteins.
Antiviral Research 11/2001; 52(1):33-41. · 4.30 Impact Factor
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ABSTRACT: Exposure of Semliki Forest virus 1 to mildly acidic conditions results in conformational changes of the viral spike proteins, which in turn leads to a pore formation across its membrane. The ability to form a pore has been ascribed to the ectodomain of the Semliki Forest virus (SFV) E1 spike protein. To elucidate whether the E1 protein per se is sufficient for low pH-dependent pore formation, we expressed E1 in Escherichia coli in an inducible manner using the pET11c expression system. The data obtained clearly showed that the E1 protein was expressed in the bacterial cell membrane and that exposure of E. coli expressing the SFV E1 protein to low pH (<6.2) resulted in a permeability change of the membrane. Thus, we conclude that the E1 protein of SFV per se is sufficient to promote pore formation under mildly acidic conditions.
Journal of Biological Chemistry 05/2001; 276(18):15453-7. · 4.77 Impact Factor
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ABSTRACT: Viruses, among them parvovirus B19 and other small, nonenveloped viruses, may be present in human blood and may contaminate plasma-derived therapeutics. Efficient inactivation or removal of such viruses, especially parvoviruses, represents a current problem and corresponding technologies are under investigation. In this report, such a technology is described.
A recently developed pasteurization of human apolipoprotein A-I (apoA-I), which is performed at 60 degrees C for 10 hours in the presence of guanidine hydrochloride (GdnHCl), was validated by using a series of model viruses, including members of the families parvoviridae and picornaviridae. The model viruses were spiked into the apoA-I- and GdnHCl-containing solutions, and virus inactivation was evaluated by infectivity assays in cell cultures. The mechanism of virus inactivation was studied by virus sedimentation analysis using the picornavirus model.
All viruses tested were inactivated to levels below the limit of detection, although different inactivation kinetics were obtained for the different viruses. The mechanism of virus inactivation by this pasteurization was disassembly of the virus particles into single proteins or small noninfectious viral subunits.
The pasteurization validated in this report has the potential to inactivate a wide range of transfusion-relevant viruses including parvoviruses and picornaviruses.
Transfusion 04/2001; 41(3):382-9. · 3.22 Impact Factor
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ABSTRACT: During the initial stages of Semliki Forest virus (SFV) infection in mammalian (baby hamster kidney, BHK) cells, the cleavage of SFV capsid protein could be detected. Analysis of subcellular fractions from SFV-infected BHK cells showed that (a) cleavage of the capsid protein occurred within a prelysosomal compartment of the endocytotic pathway, and (b) following release of the nucleocapsid into the cytoplasm, a 17.5 kD capsid protein fragment could be detected in the subcellular fraction which contained ribosomes. We have previously reported the cleavage of incoming SFV capsid protein in mosquito cells, too. Thus, the proteolytic cleavage of incoming SFV capsid protein is a feature which is common to both invertebrate and mammalian cells. These data further support our hypothesis that the cleavage of incoming capsid protein might provide the conformational change which primes the SFV nucleocapsid for uncoating.
Archives of Virology 02/1997; 142(9):1895-902. · 2.11 Impact Factor
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ABSTRACT: Exposure of Aedes albopictus cells infected with Semliki Forest virus (SFV; Togaviridae) to mildly acidic pH (5.6) results in a dramatic increase in the host cell membrane permeability due to pore formation by the virus spike proteins. Identical results were obtained when the cells were infected with two other viruses, Sindbis virus (SIN, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). This permeability change could also be observed on isolated virions of SFV, SIN and VSV by measuring the influx of propidium iodide, a nucleic acid-specific fluorescent marker, into the virions. This influx was dependent on the presence of the ectodomains of the viral spikes and could be hampered by zinc ions. Furthermore, haemagglutinin, a membrane protein of influenza A virus (Orthomyxoviridae), expressed in Aedes cells induced a change in membrane permeability identical to that induced by the spike proteins of SFV, SIN and VSV when exposed to low pH. Thus acid-induced membrane permeability changes produced by spike proteins of three different virus families could be demonstrated in infected cells as well as in virions. Therefore, the low pH-induced pore formation by viral spike proteins seems to be more than an event specific for togaviruses and might well be an inherent property of enveloped viruses that use the endocytotic pathway to infect a cell.
Journal of General Virology 01/1997; 77 ( Pt 12):3025-32. · 3.36 Impact Factor
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ABSTRACT: IgG preparations have rarely transmitted infectious diseases; however, because such transmission has occurred a few times, manufacturers are required to present experimental proof that their specific production process removes and/or inactivates viruses that may be present in the starting material.
The kinetics of virus inactivation mediated by pepsin treatment at pH 4 during the production of intravenous immunoglobulin was assessed with spiking experiments using human immunodeficiency virus, bovine viral diarrhea virus, Semliki Forest virus, and pseudorabies virus. The influence of various factors on the rate of virus inactivation also was studied by modifying the composition of the IgG solutions with respect to IgG, sucrose, and NaCl content.
Virus inactivation at 37 degrees C was extremely rapid and resulted in a complete loss of infectivity within 5 minutes to 1 hour. Inactivation was much slower at lower temperatures. Furthermore, inactivation was dependent on the solute composition. Increasing the sucrose content from 0 to 15 percent reduced the rate of inactivation of pseudorabies virus but did not affect the rate of inactivation of Semliki Forest virus. In contrast, increasing the NaCl content from 0 to 150 mM resulted in a reduction in the rate of inactivation of Semliki Forest virus, whereas the rate of inactivation of pseudorabies virus remained unaffected. Moreover, increasing the IgG concentration from 0 to 10 percent resulted in an increased rate of inactivation of pseudorabies virus but a decreased rate of inactivation of Semliki Forest virus.
Inactivation of viruses by pepsin treatment at pH 4 essentially is temperature-dependent, and the reaction rate is selectively influenced by the solute composition of the IgG solution. This has to be taken into account when safety data for different products are compared.
Transfusion 11/1996; 36(10):866-72. · 3.22 Impact Factor
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ABSTRACT: Insect cells (Aedes albopictus, clone C6/36) were infected with various variants of Semliki Forest virus including the wild type using the SFV replicon system. The variants included deletion mutants lacking one of the structural proteins and a mutant with a point mutation in p62 (SQL). The latter mutation results in a failure to process p62 to E2 and E3. After infection of the cells with different variant viruses and subsequent expression of viral proteins in the host cell plasma membrane low pH-induced pore formation was detected by measuring the efflux of a radiolabeled compound. The results of these experiments clearly showed that the E1 protein is mandatory for the acid-induced pore formation. A participation of the 6K or C-protein could be excluded. Furthermore, results obtained with the SQL mutant suggest that dissociation of the E1/E2 heterodimer and subsequent homooligomerization of E1 are required for pore formation.
Virology 07/1996; 220(1):204-7. · 3.35 Impact Factor
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ABSTRACT: The fate of Semliki Forest virus (SFV) nucleocapsid, especially the capsid protein (C-protein), was investigated during the early stages of a productive infection in mosquito Aedes albopictus cells. Infection of the cells resulted in a time dependent accumulation of a C-protein derived fragment. This fragmentation of incoming viral nucleocapsid was prevented by NH4Cl, an agent generally used to elevate the pH in acidic intracellular compartments, suggesting that a low intravesicular pH is required for this process. Density gradient analysis of the postnuclear cell lysate demonstrated that the fragmentation was associated with a cellular compartment showing a density of 1.14 +/- 0.02 g/ml. This cellular compartment was devoid from a lysosomal marker enzyme and represented the timely preceding cellular fraction through which SFV passed before encountering a lysosomal fraction. Furthermore, the intracellular distribution of the viral, 3H-uridine-labeled RNA suggested that the same fraction might represent a key cellular compartment in which the separation of the viral RNA from the viral structural proteins is primed. In conclusion, these data lead to the suggestion that the fragmentation of incoming SFV nucleocapsids in Aedes albopictus cells might be the part of the mechanism leading to the release of viral RNA into the cytosol during early stages of productive infection.
Archives of Virology 02/1996; 141(10):1805-21. · 2.11 Impact Factor
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ABSTRACT: Pore formation at mildly acidic pH by SFV spike proteins was investigated using isolated and modified virions. Modification of the virions was performed by limited proteolysis in presence of octylglucoside and resulted in the formation of E1 particles and spikeless particles, respectively. Pore formation was detected by measuring the influx of propidium iodide into the viral particles. The results obtained clearly showed that the presence of E1 alone is sufficient to promote pore formation at mildly acidic pH. Thus E1 represents the pore forming element of the viral spike proteins.
FEBS Letters 12/1995; 375(1-2):134-6. · 3.54 Impact Factor
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Biochemical Journal 10/1994; 302 ( Pt 2):313-20. · 4.90 Impact Factor
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ABSTRACT: Low amounts of Semliki Forest virus capsid protein transferred into target cells by electroporation-mediated delivery (10(3)-10(4) molecules incorporated/cell) confer thermal resistance resulting in enhanced survival. Furthermore, when exposed to 43 degrees C, these cells display an enhanced expression of heat-shock protein-70 and a translational thermotolerance. Similarly, low amounts of capsid protein transferred into cells in which transcription is blocked by actinomycin D, also protect the translational machinery at 43 degrees C. In a cell-free translation system, added capsid protein appears to modulate translational efficiency of endogenous mRNAs. At approximately 1 molecule/ribosome, capsid protein is able to enhance translation at 30 degrees C and at 43 degrees C. In contrast, high concentrations of capsid protein are responsible for a marked inhibition of protein synthesis at 30 degrees C, but only hamper translational thermotolerance at 43 degrees C. Our results favor the hypothesis that small amounts of capsid protein trigger a chaperone-like activity that is able to protect the translational machinery from thermal damage.
European Journal of Biochemistry 09/1994; 223(3):791-7. · 3.58 Impact Factor
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ABSTRACT: Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243-255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.
Bioscience Reports 01/1994; 13(6):333-47. · 2.38 Impact Factor
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ABSTRACT: Insect cells (Aedes albopictus) were infected with Semliki Forest Virus. Cell-cell fusion was then induced by lowering the extracellular pH. The underlying processes were examined by monitoring the intercellular current flow, Ij. Experimentally, this involved the use of cell pairs in conjunction with a dual voltage-clamp method. This approach allowed us to monitor the kinetics of fusion at high temporal resolution. The fusion process began shortly after acidification (delay: 3-138 sec). Initially, Ij increased in a stepwise manner, later on it developed more gradually. Fusion between two cells reached a steady state within 7-70 sec. The steps in Ij are attributable to the formation of cytoplasmic connections between the cells, presumably involving proteinaceous fusion pores. The mean amplitude of Ij steps corresponds to a conductance of 300 pS, consistent with a pore radius of 1 nm. Cytoplasmic connections developed rapidly, i.e., Ij steps occurred within less than 1 msec. The absence of Ij flickering implies that formation of cytoplasmic connections, and hence SFV induced cell-cell fusion, is irreversible.
Virology 11/1993; 196(2):541-7. · 3.35 Impact Factor
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ABSTRACT: The plasma membrane properties of Semliki forest virus-infected Aedes cells were studied using whole-cell patch-clamp recording. Cells exhibited a marked increase in membrane conductance, gm (from 0.48 +/- 0.09 nS to 14.2 +/- 10.8 nS) upon exposure to acidic pH (5.6). The membrane potential depolarized from -40.9 +/- 5.2 mV to -2.43 +/- 7.14 mV under these conditions. In uninfected cells, there was no change in gm after lowering the pH. This implies that viral fusion proteins are involved in changing the membrane properties at low pH. The increased gm in infected cells at low pH was not persistent but declined within minutes. Millimolar concentrations of calcium and zinc prevented or reverted the increase in gm. The results suggest that viral proteins, if brought to their fusogenic conformation by exposure to low pH, form unspecific pores in the residing membrane. These pores might play a role in virus entry.
Virology 04/1993; 193(1):296-302. · 3.35 Impact Factor
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ABSTRACT: The effect of dextran sulfate on the fusion of a series of enveloped viruses, bearing specifically different fusion proteins, was investigated. The fusion with model- and with biological membranes was monitored by an R18 fluorescence-dequenching fusion assay. Dextran sulfate strongly suppresses the fusion of orthomxyo- (influenza A (H1N1 and H3N2 subtypes) and influenza B), of toga- (Semliki Forest virus), and of rhabdoviruses (vesicular stomatitis and rabies virus). The fusion of the paramyxo-viruses Sendai and mumps was not significantly affected by the anionic polysaccharide. The response to dextran sulfate was virus-specific, and identical for the different members of one virusfamily, bearing the same fusion protein. It was shown that dextran sulfate attaches with high affinity to the viruses studied, but not to erythrocytes. The anionic polymer appears to attach to the fusion epitope of the viral membrane. The inhibition of virus replication in vitro shows a remarkable correlation with the observed anti-fusion effects of dextran sulfate.
Archives of Virology 02/1993; 130(3-4):317-26. · 2.11 Impact Factor
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ABSTRACT: The action of arachidonic acid and other fatty acids on membrane potential in PC12 and bovine chromaffin cells was investigated using a membrane potential-sensitive fluorescent dye. Arachidonic acid (1-40 microM) provoked dose-dependent membrane hyperpolarization, thereby reducing hyperpolarization induced by the K(+)-selective ionophore valinomycin. Other cis-unsaturated fatty acids, but not lipoxygenase products or the saturated fatty acid palmitic acid, also affected membrane potential. Tetraethylammonium blocked the arachidonic acid-induced hyperpolarization. These data suggest that cis-unsaturated fatty acids alter membrane potential in PC12 and bovine chromaffin cells by modulating K+ conductances. Valinomycin-generated hyperpolarization had no effect on agonist-induced Ca2+ influx into bovine chromaffin cells, whereas preincubation with arachidonic acid and other cis-unsaturated fatty acids blocked Ca2+ influx and secretion. We propose a model where internally generated fatty acids act as a feedback to desensitize the stimulated cell via inhibition of receptor-dependent Ca2+ influx and induction of membrane hyperpolarization.
Journal of Neurochemistry 02/1993; 60(1):282-8. · 4.06 Impact Factor
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ABSTRACT: Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used study simultaneously the diffusional and electrical properties of intercellular junctions. Diffusion studies involved injection of fluorescent molecules into one cell of a cell pair and visual inspection of their intercellular redistribution. Electrical measurements involved a dual voltage clamp method and whole-cell recording with patch pipette. The voltage clamp protocol was aimed at examining the dependency of the junctional conductance, gj, on membrane potential, Vm. Cell pairs exhibiting a voltage-dependent gj were found to allow intercellular diffusion of Lucifer Yellow CH (molecular mass, 443 Da), but not of FITC-dextran (molecular mass, 4,400 Da). This response pattern is consistent with the presence of gap junctions in the intercellular junctions. Cell pairs showing no voltage dependence of gj were found to permit intercellular diffusion of both Lucifer Yellow CH and FITC-dextran (dextran labelled with fluorescein isothiocyanate). This behaviour is compatible with the presence of cytoplasmic bridges connecting the two adjacent cells. Hence, in culture the cells investigated express two kinds of intercellular structures, gap junctions and cytoplasmic bridges.
Experimental Physiology 12/1992; 77(6):903-11. · 3.21 Impact Factor
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ABSTRACT: The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (less than 6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influx of protons into the cells (Kempf et al. Biosci. Rep. 7, 761-769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (M(r) less than 1000) was observed when infected cells were exposed to a pH less than 6.2. The pH dependence of the permeability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.
Bioscience Reports 07/1992; 12(3):221-36. · 2.38 Impact Factor
