Background and Objectives A new 10% liquid human intravenous immunoglobulin (US trade name: Gammagard Liquid; European trade name: KIOVIG) manufactured by a process with three dedicated pathogen inactivation/removal steps (solvent/detergent treatment, 35‐nm nanofiltration and low pH/elevated temperature incubation) was developed. The ability of the manufacturing process to inactivate/remove viruses and prions was investigated.Materials and Methods Virus and prion removal capacities were assessed with down‐scale spiking experiments, validated for equivalence to the large‐scale process.Results Lipid‐enveloped viruses were completely inactivated/removed by each of the three dedicated virus clearance steps, and for human immunodeficiency virus 1 (HIV‐1) and pseudorabies virus (PRV), also by the upstream cold ethanol fractionation step. Relevant non‐enveloped viruses [i.e. hepatitis A virus (HAV) and parvovirus B19 (B19V)] were effectively removed by nanofiltration and the cold ethanol fractionation step, and partial inactivation of non‐enveloped viruses was achieved by low pH incubation. Overall log reduction factors were > 20·0 for HIV‐1, > 18·1 for bovine viral diarrhoea virus, > 16·3 for West Nile virus, > 10·0 for influenza A virus subtype H5N1, > 21·8 for PRV, 12·0 for HAV, > 12·1 for encephalomyocarditis virus, 10·6 for B19V and 10·3 for mice minute virus. Prions (Western blot assay) were completely removed (≥ 3·2 mean log reduction) by a step of the cold ethanol fractionation process.Conclusions Introducing three dedicated virus‐clearance steps in the manufacturing process of immunoglobulins from human plasma provides high margins of safety.

BACKGROUND: The ever-increasing number of human H5N1 influenza virus infections may enable these viruses to acquire the ability to spread effectively among humans and potentially to cause a pandemic. Recently, more systemic virus dissemination was reported during H5N1 virus infection of humans, resulting in significant virus concentrations also in the blood. The observation has raised concerns about the safety of labile blood products for transfusion and consequentially also for plasma derivatives. To confirm the safety margins of plasma products, dedicated virus inactivation processes used during their production were investigated for their effectiveness in inactivating this virus of recent concern.STUDY DESIGN AND METHODS: Virus inactivation by steps commonly used during the manufacture of plasma derivatives, such as pasteurization for human albumin, solvent/detergent treatment for intravenous immunoglobulin (IVIG), vapor heating for factor VIII inhibitor bypassing activity, and incubation at low pH for IVIG, were investigated with a reassortant strain of H5N1 influenza virus.RESULTS: The results show that H5N1 influenza behaves as expected for lipid-enveloped viruses; that is, the virus is effectively inactivated by all the commonly used virus inactivation procedures tested.CONCLUSION: The safety margins of plasma derivatives against the theoretical transmission of H5N1 influenza virus are very substantial.

BACKGROUND: Filters with nominal pore sizes in the nanometer range are well‐established tools for enhancing the virus safety margins of plasma‐derived products, yet intrinsically less successful for smaller viruses such as hepatitis A virus (HAV) and human parvovirus B19 (B19V). The formation of virus‐antibody complexes increases the effective size of these smaller viruses and would thus improve their removal by nanofiltration. While the principle of virus removal by antibody‐dependent nanofiltration has been demonstrated with animal antisera and viruses spiked into human plasma product intermediates, the significance of these results remains unclear due to the potential contributions of xenoanti‐bodies and/or heteroagglutination in such heterologous systems.STUDY DESIGN AND METHODS: The current study investigated antibody‐dependent virus removal by nanofiltration in a heterologous animal parvovirus system to establish the concentration dependence of the effect. In addition, the phenomenon was investigated in a homologous system with custom‐made HAV and B19V antibody–free and ‐containing human immunoglobulin intermediates. Viruses were analyzed with infectivity assays and fully validated polymerase chain reaction assays that also circumvent the obscuring effects of neutralizing antibodies with infectivity assays.RESULTS: By use of the heterologous mice minute virus and the homologous HAV and B19V systems, viruses passed the 35‐nm (Planova 35N) filter in the absence of specific antibodies. Beyond a threshold virus antibody concentration, nanofiltration resulted in effective virus removal of viruses smaller than the nominal pore size of the filter used.CONCLUSION: HAV and B19V are effectively removed by antibody‐dependent 35N nanofiltration, already at intermediate antibody concentrations well below those comparable to human plasma pools for fractionation.

As a consequence of the September 2001 terrorist events, programs to protect against further such acts including potentially the use of biological warfare agents have been launched in the USA and elsewhere. As part of these initiatives, Vaccinia virus was procured for the pre-emptive vaccination of key personnel against smallpox as well as population-wide protection after an eventual exposure. The introduction of this live virus into a population at a relatively large scale represents a theoretical challenge for the safety of the blood supply, and potentially for plasma for fractionation. To strengthen further the demonstration of safety margins for plasma derived products against Vaccinia virus, the capacity of sterile filtration procedures to remove the virus was investigated. An infectivity assay for the Vaccinia virus strain which represents the majority of smallpox vaccine stocks available currently was used to investigate the potential removal of this virus by sterile filtration processes during the manufacture of plasma derivatives. Vaccinia virus behaves as predicted based on its size, i.e., an artificially added virus load is removed about 10,000-fold by the sterile filtration procedures tested. As the current investigation covered a range of different protein concentrations, filter materials and filters from different manufacturers, the results obtained are considered to be widely applicable. The current investigation supports further the high safety margins of plasma derivatives against any potential Vaccinia virus content of plasma for fractionation. As the large size is a general feature of Orthopox viruses, the results would also provide assurance against poxviruses identified more recently, for example, Monkeypox virus.

The genome of hepatitis B virus (HBV) encodes two transcriptional activators: the HBx protein and the PreS2-activator large surface protein (LHBs). Both proteins trigger activation of c-Raf-1/MEK kinase cascade. In case of HBx this can be mediated by a PKC-independent and Ras-dependent mechanism, in case of LHBs activation is PKC-dependent and does not require Ras. Selective destruction of either LHBs- or of HBx-specific activation does not result in significant decrease of viral production from transfected HepG2 cells. Simultaneous inhibition of LHBs- and HBx-dependent activation by blocking signaling steps common to both activators, using trans dominant negative c-Raf-1- or MEK-specific inhibitors, abolished HBV gene expression. In accordance with this no HBV propagation was observed after transfection of a mutated HBV genome defective for HBx- and PreS2-activator function. A detailed analysis revealed that the observed inhibition of HBV- propagation is because of a significant reduction of HBV-specific RNA resulting in an inhibition of the de novo synthesis of viral compounds (viral proteins and nucleic acid) and not by blocking secretion or assembly of the virus. Based on these results we conclude that transcriptional-activator function, mediated by the c-Raf-1/MEK signaling cascade, is essential for HBV gene expression.

BACKGROUND:During the 2002 West Nile virus (WNV) epidemic in the US, virus transmission through solid organ transplantation and transfusion of blood components was observed. This raised concerns about the safety of plasma derivatives. To verify the safety margins of these products, which were initially shown with a panel of model viruses including some very similar to WNV, the effectiveness of the virus inactivation procedures incorporated into their manufacturing processes was reinvestigated. STUDY DESIGN AND METHODS:An infectivity assay for 1999 New York isolate of WNV was established to investigate virus inactivation steps commonly used during the manufacture of plasma derivatives, such as pasteurization for human albumin, S/D treatment for IVIG and FVIII, vapor heating for FVIII inhibitor-bypassing activity, and incubation at low pH for IVIG. RESULTS:The results show that WNV behaves exactly as had been predicted based on available data for similar model viruses; that is, it is readily inactivated by all the commonly used virus inactivation procedures tested. CONCLUSION:Our investigation verifies the safety margins of plasma derivatives against a potential transmission of WNV and that the model virus concept is valid for predicting the behavior of closely related viruses.

The percentage of cells successfully transformed by Agrobacteria is usually very low (not more than 10% and usually much less; Thomas et al., 1989). In this report we show that in carrot (Daucus carota L., ssp. sativus) cell suspensions transformation efficiency was strongly improved by using cell cycle synchronized cells. Fluorodesoxyuridine (FDU) was added for 24 h to inhibit thymidine synthesis. This blocked the cell cycle at the transition from G1- to S-phase. Then the block was released by applying thymidine. A high rate of transformation was obtained when Agrobacterium tumefaciens was added concurrently with thymidine. As examples of efficient and long term foreign gene expression in transgenic cells, the reporter enzyme β-glucuronidase (GUS) as a model as well as the major hepatitis B virus surface protein were used. Both genes were linked to the MAS promoter. In carrot cell suspensions containing the viral gene, the corresponding viral protein was produced. In roots of mature transgenic carrot plants generated through somatic embryogenesis and raised in soil as well as in callus cultures derived thereof, the viral protein was also produced.

In chronically infected patients, hepatitis B virus (HBV) particles reach numbers as large as >109 genome equivalents (GE)/ml of serum. However, expression of infectious HBV particles in cell culture only yields 105–106 GE/ml, which is insufficient for many studies. HBV transcription and possibly replication is dependent on hepatocyte-specific differentiation. Thus, we tested several cell culture parameters that have been reported to enhance the expression of hepatocyte-specific markers, such as growth on different extracellular matrices, different cell culture media, low concentrations of fetal calf serum (FCS) and the addition of dimethyl sulfoxide (DMSO) to the medium. Lower concentrations of FCS, growth on collagen and inclusion of DMSO in the medium only moderately enhanced HBV production in vitro when applied individually. However, combinations of these parameters optimised cell culture conditions and reproducibly increased the release of HBV particles about 100-fold to titres >108 GE/ml of culture medium.

Entry of enveloped viruses is often mediated by an aminoterminal hydrophobic fusion peptide of a viral surface protein. The S domain of the hepatitis B virus surface protein contains a putative fusion peptide at position 7-18, but no systems are available to study its function directly. We tested the functionality of this peptide and a related peptide from another hepadnavirus in the context of the well-characterized influenza virus hemagglutinin H7 using gene mutation. The chimeric hemagglutinins could be expressed stably in CV 1 cells and were transported to the cell surface. The chimeras were incompletely cleaved by cellular proteases but cleavage could be completed by trypsin treatment of the cells. The chimeras did not differ in receptor binding, i.e. erythrocyte binding. Hemifusion and fusion pore formation were detected with membrane or cytosolic fluorescent dye-labeled erythrocytes as target structures of the hemagglutinin. Five of six different chimeras mediated hemifusion in 20-54% of the hemagglutinin-expressing cells, complete fusion and syncytium formation was not observed. The data suggest that the sequence 7-18 of the hepatitis B S domain may indeed initiate the first step of viral entry, i.e. hemifusion.

Hepatitis B surface proteins play a central role in the assembly of the virus and in the infection of the host cells. Whereas some functional aspects of the proteins have been studied in detail, little is known about their structure. Since X-ray analysis of these proteins appear unlikely in the near future, we chose to use a variety of computer-aided methods to improve the model for the major surface protein (SHBs). We here describe the model, discuss it in light of current results in the literature and discuss new functional implications of SHBs.