Biological Safety of a Highly Purified 10% Liquid Intravenous Immunoglobulin Preparation from Human Plasma.

BACKGROUND: A highly purified 10% liquid intravenous immunoglobulin, IQYMUNE®, has been developed using an innovative manufacturing process including an affinity chromatography step for the removal of anti-A and anti-B hemagglutinins. OBJECTIVES: The pathogen (viruses and prions) clearance efficacy of the manufacturing process and its robustness for critical steps were investigated. METHODS: The manufacturing process of IQYMUNE® includes two dedicated complementary virus reduction steps: solvent/detergent (S/D) treatment and 20 nm nanofiltration as well as two contributing steps, namely caprylic acid fractionation and anion-exchange chromatography. The clearance capacity and robustness of these steps were evaluated with a wide range of viruses (enveloped and non-enveloped) and with a model of human transmissible spongiform encephalopathies (TSEs). RESULTS: The IQYMUNE® manufacturing process demonstrated a high and robust virus removal capacity with global reduction factors (RFs) of relevant and model viruses: ≥14.8 log10 for human immunodeficiency virus type 1 (HIV-1), ≥16.9 log10 for bovine viral diarrhoea virus (BVDV)/Sindbis virus, ≥15.7 log10 for pseudorabies virus (PRV), ≥12.8 log10 for encephalomyocarditis virus (EMCV) and 11.0 log10 for porcine parvovirus (PPV). The process also exhibited a high removal capacity for the TSE agent with an overall RF of ≥12.9 log10 due to the complementary actions of the caprylic acid fractionation, anion-exchange chromatography and nanofiltration steps. CONCLUSION: Data from virus and prion clearance studies fully support the high safety profile of IQYMUNE®, with a minimal reduction of 11 log10 for the smallest and most resistant non-enveloped virus, PPV, and more than 12 log10 for the TSE agent.

Role of Prion Replication in the Strain-dependent Brain Regional Distribution of Prions.

One intriguing feature of prion diseases is their strain variation. Prion strains are differentiated by the clinical consequences they generate in the host, their biochemical properties, and their potential to infect other animal species. The selective targeting of these agents to specific brain structures have been extensively used to characterize prion strains. However, the molecular basis dictating strain-specific neurotropism are still elusive. In this study, isolated brain structures from animals infected with four hamster prion strains (HY, DY, 139H, and SSLOW) were analyzed for their content of protease-resistant PrP(Sc) Our data show that these strains have different profiles of PrP deposition along the brain. These patterns of accumulation, which were independent of regional PrP(C) production, were not reproduced by in vitro replication when different brain regions were used as substrate for the misfolding-amplification reaction. On the contrary, our results show that in vitro replication efficiency depended exclusively on the amount of PrP(C) present in each part of the brain. Our results suggest that the variable regional distribution of PrP(Sc) in distinct strains is not determined by differences on prion formation, but on other factors or cellular pathways. Our findings may contribute to understand the molecular mechanisms of prion pathogenesis and strain diversity.

Dissociation of prion protein amyloid seeding from transmission of a spongiform encephalopathy.

Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar "prion-like" mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer's disease. We investigated if misfolding and aggregation of corrupted prion protein (PrP(TSE)) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrP(TSE) from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrP(TSE). In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrP(TSE) is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrP(TSE) in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.

Post-translational changes to PrP alter transmissible spongiform encephalopathy strain properties.

The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrP(Sc), an abnormal conformer of the host glycoprotein PrP(C). TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrP(Sc) with the same amino-acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post-translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild-type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N-glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild-type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain-specific characteristics of the 79A TSE strain changed when PrP(Sc) was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post-translational changes to PrP which we propose result in the selection of mutant TSE strains.

Co-infection with the friend retrovirus and mouse scrapie does not alter prion disease pathogenesis in susceptible mice.

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.

Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models.

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.

Gene knockout of tau expression does not contribute to the pathogenesis of prion disease.

Prion diseases or transmissible spongiform encephalopathies are a group of fatal and transmissible disorders affecting the central nervous system of humans and animals. The principal agent of prion disease transmission and pathogenesis is proposed to be an abnormal protease-resistant isoform of the normal cellular prion protein. The microtubule-associated protein tau is elevated in patients with Creutzfeldt-Jakob disease. To determine whether tau expression contributes to prion disease pathogenesis, tau knockout and control wild-type mice were infected with the M1000 strain of mouse-adapted human prions. Immunohistochemical analysis for total tau expression in prion-infected wild-type mice indicated tau aggregation in the cytoplasm of a subpopulation of neurons in regions associated with spongiform change. Western immunoblot analysis of brain homogenates revealed a decrease in total tau immunoreactivity and epitope-specific changes in tau phosphorylation. No significant difference in incubation period or other disease features were observed between tau knockout and wild-type mice with clinical prion disease. These results demonstrate that, in this model of prion disease, tau does not contribute to the pathogenesis of prion disease and that changes in the tau protein profile observed in mice with clinical prion disease occurs as a consequence of the prion-induced pathogenesis.

Spongiform degeneration induced by neuropathogenic murine coronavirus infection.

Soluble receptor-resistant mutant 7 (ssr7) is isolated from a highly neurovirulent mouse hepatitis virus (MHV) JHMV cl-2 strain (cl-2). srr7 exhibits lower virulence than its maternal strain in infected mice, which is typically manifested in a longer lifespan. In this study, during the course of infection with srr7, small spongiotic lesions became apparent at 2 days post-inoculation (pi), they spread out to form spongiform encephalopathy by 8 to 10 days pi. We recently reported that the initial expressions of viral antigens in the brain are detected in the infiltrating monocyte lineage and in ependymal cells. Here, we demonstrate that the next viral spread was observed in glial fibrillary acidic protein-positive cells or nestin-positive progenitor cells which take up positions in the subventricular zone (SVZ). From this restricted site of infection in the SVZ, a large area of gliosis extended deep into the brain parenchyma where no viral antigens were detected but vacuolar degeneration started at 48 h pi of the virus. The extremely short incubation period compared with other experimental models of infectious spongiform degeneration in the brain would provide a superior experimental model to investigate the mechanism of spongiotic lesions formation.

Identification of atypical scrapie in Canadian sheep.

Scrapie, a transmissible spongiform encephalopathy of sheep and goats, exists in most small ruminant-producing countries of the world. A novel form of this disease was recently recognized and is known by various names, including Nor98, Nor98-like, and atypical scrapie. Differing from classic scrapie in epidemiology, histopathology, and biochemical characteristics, atypical scrapie cases have been identified throughout Europe and in the United States. Enhanced scrapie surveillance efforts recently identified 3 cases of atypical scrapie in Canada.

Systemic challenge with the TLR3 agonist poly I:C induces amplified IFNalpha/beta and IL-1beta responses in the diseased brain and exacerbates chronic neurodegeneration.

The role of inflammation in the progression of neurodegenerative disease remains unclear. We have shown that systemic bacterial insults accelerate disease progression in animals and in patients with Alzheimer's disease. Disease exacerbation is associated with exaggerated CNS inflammatory responses to systemic inflammation mediated by microglia that become 'primed' by the underlying neurodegeneration. The impact of systemic viral insults on existing neurodegenerative disease has not been investigated. Polyinosinic:polycytidylic acid (poly I:C) is a toll-like receptor-3 (TLR3) agonist and induces type I interferons, thus mimicking inflammatory responses to systemic viral infection. In the current study we hypothesized that systemic challenge with poly I:C, during chronic neurodegenerative disease, would amplify CNS inflammation and exacerbate disease. Using the ME7 model of prion disease and systemic challenge with poly I:C (12 mg/kg i.p.) we have shown an amplified expression of IFN-alpha and beta and of the pro-inflammatory genes IL-1beta and IL-6. Similarly amplified expression of specific IFN-dependent genes confirmed that type I IFNs were secreted and active in the brain and this appeared to have anti-inflammatory consequences. However, prion-diseased animals were susceptible to heightened acute sickness behaviour and acute neurological impairments in response to poly I:C and this treatment also accelerated disease progression in diseased animals without effect in normal animals. Increased apoptosis coupled with double-stranded RNA-dependent protein kinase (PKR) and Fas transcription suggested activation of interferon-dependent, pro-apoptotic pathways in the brain of ME7+poly I:C animals. That systemic poly I:C accelerates neurodegeneration has implications for the control of systemic viral infection during chronic neurodegeneration and indicates that type I interferon responses in the brain merit further study.

Elimination capacity of a TSE-model agent in the manufacturing process of Alphanate/Fanhdi, a human factor VIII/VWF complex concentrate.

The variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy (TSE), mainly present in the UK and is associated with the ingestion of bovine products affected with bovine spongiform encephalopathy. Manufacturers of biological products must investigate the ability of their production processes to remove TSE agents. We studied the purification steps in the manufacturing process of two FVIII/VWF concentrates (Alphanate) and Fanhdi in their ability to eliminate an experimental TSE-model agent. Hamster scrapie strain 263K brain-derived materials were spiked into samples of the solutions taken before various stages during its production: 3.5% polyethylene glycol (PEG) precipitation, heparin affinity chromatography and saline precipitation/final filtrations. PEG precipitation and affinity chromatography were studied both as isolated and combined steps. TSE agent removal was determined using a laboratory scale model representative of the industrial manufacturing process. The prion protein (PrP(Sc)) was measured with Western blot and TSE infectivity was measured with bioassay. Western blot results were in agreement with those obtained by bioassay, showing a significant removal capacity in the production process: 3.21-3.43 log(10) for the PEG precipitation; about 3.45 log(10) for the affinity chromatography; and around 2.0 log(10) for the saline precipitation plus final filtrations. PEG precipitation and heparin affinity chromatography were demonstrated to be two complementary TSE-model agent removal mechanisms with total removal being the sum of the two. An overall reduction factor of around 8 log(10) can be deduced. The tests from the production process of FVIII/VWF complex concentrates have demonstrated their potential for eliminating TSE agents.

Retinal function and morphology are altered in cattle infected with the prion disease transmissible mink encephalopathy.

Transmissible spongiform encephalopathies (TSEs) are a group of diseases that result in progressive and invariably fatal neurologic disease in both animals and humans. TSEs are characterized by the accumulation of an abnormal protease-resistant form of the prion protein in the central nervous system. Transmission of infectious TSEs is believed to occur via ingestion of prion protein-contaminated material. This material is also involved in the transmission of bovine spongiform encephalopathy ("mad cow disease") to humans, which resulted in the variant form of Creutzfeldt-Jakob disease. Abnormal prion protein has been reported in the retina of TSE-affected cattle, but despite these observations, the specific effect of abnormal prion protein on retinal morphology and function has not been assessed. The objective of this study was to identify and characterize potential functional and morphologic abnormalities in the retinas of cattle infected with a bovine-adapted isolate of transmissible mink encephalopathy. We used electroretinography and immunohistochemistry to examine retinas from 10 noninoculated and 5 transmissible mink encephalopathy-inoculated adult Holstein steers. Here we show altered retinal function, as evidenced by prolonged implicit time of the electroretinogram b-wave, in transmissible mink encephalopathy-infected cattle before the onset of clinical illness. We also demonstrate disruption of rod bipolar cell synaptic terminals, indicated by decreased immunoreactivity for the alpha isoform of protein kinase C and vesicular glutamate transporter 1, and activation of Müller glia, as evidenced by increased glial fibrillary acidic protein and glutamine synthetase expression, in the retinas of these cattle at the time of euthanasia due to clinical deterioration. This is the first study to identify both functional and morphologic alterations in the retinas of TSE-infected cattle. Our results support future efforts to focus on the retina for the development of new strategies for the diagnosis of TSEs.

Strain-specific viral properties of variant Creutzfeldt-Jakob disease (vCJD) are encoded by the agent and not by host prion protein.

Human CJD, endemic sheep scrapie, epidemic bovine spongiform encephalopathy (BSE), and other transmissible spongiform encephalopathies (TSEs), are caused by a group of related but molecularly uncharacterized infectious agents. The UK-BSE agent infected many species, including humans where it causes variant CJD (vCJD). As in most viral infections, different TSE disease phenotypes are determined by both the agent strain and the host species. TSE strains are most reliably classified by incubation time and regional neuropathology in mice expressing wild-type (wt) prion protein (PrP). We compared vCJD to other human and animal derived TSE strains in both mice and neuronal cultures expressing wt murine PrP. Primary and serial passages of the human vCJD agent, as well as the highly selected mutant 263K sheep scrapie agent, revealed profound strain-specific characteristics were encoded by the agent, not by host PrP. Prion theory posits that PrP converts itself into the infectious agent, and thus short incubations require identical PrP sequences in the donor and recipient host. However, wt PrP mice injected with human vCJD brain homogenates showed dramatically shorter primary incubation times than mice expressing only human PrP, a finding not in accord with a PrP species barrier. All mouse passage brains showed the vCJD agent derived from a stable BSE strain. Additionally, both vCJD brain and monotypic neuronal cultures produced a diagnostic 19 kDa PrP fragment previously observed only in BSE and vCJD primate brains. Monotypic cultures can be used to identify the intrinsic, strain-determining molecules of TSE infectious particles.

Prion disease transmission: can we apply standard precautions to prevent or reduce risks?

Prion diseases present unique challenges to healthcare facilities, both in the care and treatment of patients. A significant cause for concern is in the routine reprocessing of medical devices used on patients and how disease transmission can be prevented on the reuse of devices. Investigations have shown that prion disease can be transmitted on medical devices, which can be a concern given the long incubation times associated with these diseases and that guidelines to control transmission only really apply in a small number of known or at risk cases. It is only recently that medical device-associated cleaning, disinfection and sterilization technologies have been investigated and the results of these studies are summarized in this report. The evidence would suggest that many simple decontamination steps can be applied to dramatically reduce the risks to patients, but the research has also given some surprises. Overall, it is reasonable to expect that standard precautions will be able to be applied both today as well as in the future to reduce the risk of prion disease transmission as well as the many other human pathogen concerns, although this may mean changes in some of our practices.

Virus-induced alterations of membrane lipids affect the incorporation of PrP Sc into cells.

Prion diseases are fatal neurodegenerative disorders characterized by long incubation periods. To investigate whether concurrent diseases can modify the clinical outcome of prion-affected subjects, we tested the effect of viral infection on the binding and internalization of PrP(Sc), essential steps of prion propagation. To this effect, we added scrapie brain homogenate or purified PrP(Sc) to fibroblasts previously infected with minute virus of mice (MVM), a mouse parvovirus. We show here that the rate of incorporation of PrP(Sc) into MVM-infected cells was significantly higher than that observed for naïve cells. Immunostaining of cells and immunoblotting of subcellular fractions using antibodies recognizing PrP and LysoTracker, a lysosomal marker, revealed that in both control and MVM-infected cells the incorporated PrP(Sc) was associated mostly with lysosomes. Interestingly, flotation gradient analysis revealed that the majority of the PrP(Sc) internalized into MVM-infected cells shifted toward raft-containing low-density fractions. Concomitantly, the MVM-infected cells demonstrated increased levels of the glycosphingolipid GM1 (an essential raft lipid component) throughout the gradient and a shift in caveolin 1 (a raft protein marker) toward lighter membrane fractions compared with noninfected cells. Our results suggest that the effect of viral infection on membrane lipid composition may promote the incorporation of exogenous PrP(Sc) into rafts. Importantly, membrane rafts are believed to be the conversion site of PrP(C) to PrP(Sc); therefore, the association of exogenous PrP(Sc) with such membrane microdomains may facilitate prion infection.

Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles.

BACKGROUND INFORMATION: TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrP(Sc), a conformationally altered isoform of the normal prion protein (PrP(C)), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a). RESULTS: The present study shows that N2a cells infected with scrapie release the prion agent into the cell culture medium in association with exosome-like structures and viral particles of endogenous origin. We found that both prion proteins and scrapie infectivity are mainly associated with exosome-like structures that contain viral envelope glycoprotein and nucleic acids, such as RNAs. CONCLUSIONS: The dissemination of prions in N2a cell culture is mediated through the exosomal pathway.

Tubulovesicular structures are present in brains of hamsters infected with the Echigo-1 strain of Creutzfeldt-Jakob disease agent.

Tubulovesicular structures (particles; TVS) are virion-like particles 25-30 nm in diameter found by thin-section electron microscopy in brains of all prion diseases including scrapie, Creutzfeldt-Jakob disease (CJD) fatal familial insomnia (FFI) and Gerstmann-Sträussler-Scheineker disease (GSS) as well as in cell cultures infected with TSE agents. TVS are regarded as a disease-specific ultrastructural marker for TSEs and, by those not completely satisfied with the prion hypothesis, they are even considered to be a possible candidate for the infectious TSE agent itself. A caveat regarding that interpretation stemmed from previous failures to find TVS by electron microscopic studies of tissues from animals infected with the Echigo-1 strain of CJD agent. We now report detecting TVS in brains of hamsters infected with that strain of CJD agent, albeit with a very low frequency.

Imaging of slow viruses.

The symptoms associated with slow viral or prion diseases of the central nervous system tend to have multiple neurologic symptoms, and different patients may present with different symptoms. This review discusses the most common slow virus infections and their imaging findings.

Transcriptional stability of cultured cells upon prion infection.

Prion infections induce severe disruption of the central nervous system with neuronal vacuolation and extensive glial reactions, and invariably lead to death of affected individuals. The molecular underpinnings of these events are not well understood. To better define the molecular consequences of prion infections, we analyzed the transcriptional response to persistent prion infection in a panel of three murine neural cell lines in vitro. Colony spot immunochemistry assays indicated that 65-100% of cells were infected in each line. Only the Nav1 gene was marginally modulated in one cell line, whereas transcripts previously reported to be derailed in prion-infected cells were not confirmed in the present study. We attribute these discrepancies to the experimental stringency of the current study, which was performed under conditions designed to minimize potential genetic drifts. These findings are at striking variance with gene expression studies performed on whole brains upon prion infections in vivo, suggesting that many of the latter changes represent secondary reactions to infection. We conclude that, surprisingly, there are no universal transcriptional changes induced by prion infection of neural cells in vitro.