Christoph Kempf

Photodynamic inactivation of enveloped viruses using buckminsterfullerene

In a process for the inactivation of enveloped viruses in a biological fluid buckminsterfullerene (C60) is used as a photosensitizer. In a photodynamic processes singlet oxygen is generated which is the active agent for the inactivation of viruses present in the fluid. The process comprises the steps of (a) contacting of a solution or dispersion of material derived from the human body or from the bodies of animals with buckminsterfullerene as a photosensitizer, of (b) saturating the solution or dispersion with oxygen; and of (c) irradiating the solution or dispersion with visible or invisible light for activating the oxygen into the singlet state until viruses contained in the solution or dispersion are inactivated. This virus inactivation is specially suitable for protein solutions, e.g. bovine serum albumin, BSA or plasma products of human origin.

Low pH-Dependent Endosomal Processing of the Incoming Parvovirus Minute Virus of Mice Virion Leads to Externalization of the VP1 N-Terminal Sequence (N-VP1), N-VP2 Cleavage, and Uncoating of the Full-Length Genome

ABSTRACT Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.

Cytoplasmic Trafficking of Minute Virus of Mice: Low-pH Requirement, Routing to Late Endosomes, and Proteasome Interaction

ABSTRACT The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A 1 and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation.

Robustness of solvent/detergent treatment of plasma derivatives: a data collection from Plasma Protein Therapeutics Association member companies.

BACKGROUND: Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method.

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.

Different susceptibility of B19 virus and mice minute virus to low pH treatment.

BACKGROUND:  Parvoviridae are small nonenveloped viruses that are known to be highly resistant to physico‐chemical treatments. Because low pH is frequently applied to process intermediates or final products, the impact of such conditions on the human erythrovirus B19 (B19V) and the mouse parvovirus (mice minute virus, MMV) was assessed, which is often used as a model for B19V. Owing to the lack of a suitable cultivation and/or detection system for B19V no such data exist so far.

Stability of minute virus of mice against temperature and sodium hydroxide.

Treatment with steam and/or dilute NaOH are commonly used techniques to disinfect manufacturing vessels and tools in the pharmaceutical industry. The aim of this procedure is sanitisation and inactivation of microbiological and viral contaminants. Here we describe the inactivation of the mouse parvovirus Minute Virus of Mice (MVM) under these conditions. Parvoviruses are known to be resistant to physico-chemical treatment and one representative of this family, the human parvovirus B19, is a potential contaminant of blood plasma. We show inactivation kinetics for MVM treated with wet-heat (70, 80, 90 degrees C) and with 0.01-1 M NaOH solutions (pH >/=11.9). Robust inactivation was only achieved at 90 degrees C for at least 10 min and in NaOH solutions of pH >/=12.8 (0.1 M NaOH). It was observed, that aggregation of viruses might protect viral particles from inactivation by NaOH. Therefore, appropriate sample preparation of spiking material is important for accurate simulation of the naturally occurring situation. The observed stability at pH 11.8 exceeds the previously reported upper limit of pH 9. Inactivation was due to disintegration of the viral capsid as assessed by accessibility of viral DNA for endonucleases.

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.

Conformational Changes in the VP1-Unique Region of Native Human Parvovirus B19 Lead to Exposure of Internal Sequences That Play a Role in Virus Neutralization and Infectivity

ABSTRACT The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A2 (PLA2) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA2 activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA2 potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.

Electrical coupling between cells of the insect Aedes albopictus.

1. Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used to study the electrical properties of intercellular junctions. A double voltage‐clamp approach was adopted to control the voltage gradient between the cells and measure the intracellular current flow. 2. Determinations of junctional conductance (gj) revealed two types of intercellular contacts, gap junctions and cytoplasmic bridges. Identification occurred by means of functional criteria, i.e. the dependency of gj on (i) junctional membrane potential, (ii) non‐junctional membrane potential, and (iii) heptanol. 3. In cell pairs with putative gap junctions, gj was dependent on the junctional membrane potential (Vj). When determined at the beginning of voltage pulses, gj was insensitive to Vj; when determined at the end of 15 s pulses, it depended on Vj in a bell‐shaped manner (70% decrease for a change in Vj of +/‐ 75 mV). 4. These cell pairs also showed a dependency of gj on the non‐junctional membrane potential (Vm). When determined immediately after changing the non‐junctional membrane potential in both cells, gj was not affected by Vm; when determined 30 s later, gj was modified by Vm in a S‐shaped fashion (100% decrease when Vm was depolarized to +50 mV). 5. Exposure to 3 mM‐heptanol gave rise to complete and reversible block of gj in cell pairs with putative gap junctions. 6. Cell pairs susceptible to uncoupling by heptanol revealed junctional currents indicative of the operation of gap junction channels. The single‐channel conductance, determined at a Vm of ‐50 to ‐70 mV, was 133 pS. 7. In the case of putative cytoplasmic bridges, gj was insensitive to the junctional and non‐junctional membrane potential. In addition, it was not affected by 3 mM‐heptanol. 8. While most cell pairs showed functional properties characteristic of gap junctions or cytoplasmic bridges, few cell pairs exhibited junctional currents compatible with the co‐existence of both junctional structures.