6th International Immunoglobulin Symposium: poster presentations.

The posters presented at the 6th International Immunoglobulin Symposium covered a wide range of fields and included both basic science and clinical research. From the abstracts accepted for poster presentation, 12 abstracts were selected for oral presentations in three parallel sessions on immunodeficiencies, autoimmunity and basic research. The immunodeficiency presentations dealt with novel, rare class-switch recombination (CSR) deficiencies, attenuation of adverse events following IVIg treatment, association of immunoglobulin (Ig)G trough levels and protection against acute infection in patients with X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVID), and the reduction of class-switched memory B cells in patients with specific antibody deficiency (SAD). The impact of intravenous immunoglobulin on fetal alloimmune thrombocytopenia, pregnancy and postpartum-related relapses in multiple sclerosis and refractory myositis, as well as experiences with subcutaneous immunoglobulin in patients with multi-focal motor neuropathy, were the topics presented in the autoimmunity session. The interaction of dendritic cell (DC)-SIGN and alpha2,6-sialylated IgG Fc and its impact on human DCs, the enrichment of sialylated IgG in plasma-derived IgG, as wells as prion surveillance and monitoring of anti-measles titres in immunoglobulin products, were covered in the basic science session. In summary, the presentations illustrated the breadth of immunoglobulin therapy usage and highlighted the progress that is being made in diverse areas of basic and clinical research, extending our understanding of the mechanisms of immunoglobulin action and contributing to improved patient care.

Investigations of prion and virus safety of a new liquid IVIG product.

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.

Strengths and limitations of the model virus concept.

New plasma- or cell culture-based pharmaceutical manufacturing processes must be validated for their ability to eliminate potentially contaminating pathogens. To evaluate the virus elimination potential of such a process, current guidelines propose the use of model viruses. This approach is discussed based on two examples. These examples show the strengths of this approach but also its limitations. The blood processing industry was recently challenged by the emergence of a West Nile Virus (WNV) epidemic in the United States. The susceptibility of WNV and a frequently used model virus to commonly used inactivation methods is compared. Current data show a good correlation. Due to its physico-chemical properties and the high viremic titers, B19 virus (B19V), a small (diameter 18-26 nm), robust, non-enveloped parvovirus, is a considerable challenge for the plasma processing industry. Mice minute virus (MMV), an animal parvovirus, is used as a model for B19V. Data show that B19V is considerably more susceptible to some physico-chemical inactivation methods than MMV. The examples of WNV and B19V show that the model virus concept is a practicable tool to evaluate the safety of plasma- or cell culture-derived pharmaceuticals regarding known and emerging viruses. It also underlines the need for investigational studies of relevant viruses if they can be handled in a normal virology laboratory, under moderate biosafety conditions.

Virus inactivation and protein modifications by ethyleneimines.

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.

Inactivation of enveloped viruses by singlet oxygen thermally generated from a polymeric naphthalene derivative.

Inactivation of viruses can be induced by singlet oxygen generating agents. The water-insoluble polymeric compound PVNE (poly (1,4-dimethyl-6-vinylnaphthalene-1,4-endoperoxide)) is used as a storage for reactive oxygen and is able to produce thermally generated 1O2 in a dark-reaction. Enveloped viruses from two different families, Semliki Forest virus (SFV, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae) showed a loss of infectivity of up to 8 log10/ml (TCID50) when incubated at 37 degrees C with PVNE in buffered solutions. PVNE produces singlet oxygen by thermal decomposition without irradiation. Such chemically generated oxygen excludes reactions involving radicals (type I photoreactions), a problem often encountered in photodynamic processes utilizing dyes as sensitizers. In addition, the water insolubility of the oxygen-carrier allows an easy removal and recycling from aqueous solutions. Therefore, it may prove useful in the inactivation of viruses in biological systems and may be a helpful tool in studies concerning the inactivation mechanism by 1O2.

Buckminsterfullerene and photodynamic inactivation of viruses.

The development of new virus inactivation procedures has become an area of growing interest mainly due to increased demands concerning the safety of biological products. Photochemical processes represent the most promising methods for the future to inactivate viruses. In these methods, dyes are the most widely used photosensitising reagents. The current article covers a new interesting alternative, namely the use of buckminsterfullerene (C60). The unique properties of this molecule make it a valid candidate for future applications in the inactivation of viruses in biological fluids. Copyright 1998 John Wiley & Sons, Ltd.

Photodynamic inactivation of enveloped viruses by buckminsterfullerene.

Photodynamic reactions induced by singlet oxygen-generating agents are known to inactivate enveloped viruses. In this report we demonstrate that the water-insoluble photosensitizer buckminsterfullerene (C60) can be used to mediate the inactivation of enveloped viruses. Viruses from two different families, Semliki Forest virus (SFV, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae) were used as model systems. Buffered solutions containing C60 plus either of these viruses were illuminated with visible light for up to 5 h, resulting in a loss of infectivity of more than 7 log10/ml (TCID50). Furthermore, it was demonstrated that this viral inactivation was oxygen-dependent and equally efficient in solutions containing protein. C60 yields singlet oxygen in very high amounts and is completely inert to photo-oxidative destruction. In addition, it can be easily removed and recycled from aqueous solutions. For these reasons, it may prove useful in the inactivation of viruses in biological systems.

Low pH-induced pore formation by spike proteins of enveloped viruses.

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.

Identification of the pore forming element of Semliki Forest virus spikes.

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.