Transmission Of Transmissible Spongiform Encephalopathies Research Articles

The glycosylation status of PrPC is a key factor in determining transmissible spongiform encephalopathy transmission between species.

ABSTRACTThe risk of transmission of transmissible spongiform encephalopathies (TSE) between different species has been notoriously unpredictable because the mechanisms of transmission are not fully understood. A transmission barrier between species often prevents infection of a new host with a TSE agent. Nonetheless, some TSE agents are able to cross this barrier and infect new species, with devastating consequences. The host PrPC misfolds during disease pathogenesis and has a major role in controlling the transmission of agents between species, but sequence compatibility between host and agent PrPC does not fully explain host susceptibility. PrPC is posttranslationally modified by the addition of glycan moieties which have an important role in the infectious process. Here, we show in vivo that glycosylation of the host PrPC has a significant impact on the transmission of TSE between different host species. We infected mice carrying different glycosylated forms of PrPC with two human agents (sCJDMM2 and vCJD) and one hamster strain (263K). The absence of glycosylation at both or the first PrPC glycosylation site in the host results in almost complete resistance to disease. The absence of the second site of N-glycan has a dramatic effect on the barrier to transmission between host species, facilitating the transmission of sCJDMM2 to a host normally resistant to this agent. These results highlight glycosylation of PrPC as a key factor in determining the transmission efficiency of TSEs between different species.IMPORTANCE The risks of transmission of TSE between different species are difficult to predict due to a lack of knowledge over the mechanisms of disease transmission; some strains of TSE are able to cross a species barrier, while others do not. The host protein, PrPC, plays a major role in disease transmission. PrPC undergoes posttranslational glycosylation, and the addition of these glycans may play a role in disease transmission. We infected mice that express different forms of glycosylated PrPC with three different TSE agents. We demonstrate that changing the glycosylation status of the host can have profound effects on disease transmission, changing host susceptibility and incubation times. Our results show that PrPC glycosylation is a key factor in determining risks of TSE transmission between species.

Glycosylphosphatidylinositol Anchoring Directs the Assembly of Sup35NM Protein into Non-fibrillar, Membrane-bound Aggregates

In prion-infected hosts, PrPSc usually accumulates as non-fibrillar, membrane-bound aggregates. Glycosylphosphatidylinositol (GPI) anchor-directed membrane association appears to be an important factor controlling the biophysical properties of PrPSc aggregates. To determine whether GPI anchoring can similarly modulate the assembly of other amyloid-forming proteins, neuronal cell lines were generated that expressed a GPI-anchored form of a model amyloidogenic protein, the NM domain of the yeast prion protein Sup35 (Sup35(GPI)). We recently reported that GPI anchoring facilitated the induction of Sup35(GPI) prions in this system. Here, we report the ultrastructural characterization of self-propagating Sup35(GPI) aggregates of either spontaneous or induced origin. Like membrane-bound PrPSc, Sup35(GPI) aggregates resisted release from cells treated with phosphatidylinositol-specific phospholipase C. Sup35(GPI) aggregates of spontaneous origin were detergent-insoluble, protease-resistant, and self-propagating, in a manner similar to that reported for recombinant Sup35NM amyloid fibrils and induced Sup35(GPI) aggregates. However, GPI-anchored Sup35 aggregates were not stained with amyloid-binding dyes, such as Thioflavin T. This was consistent with ultrastructural analyses, which showed that the aggregates corresponded to dense cell surface accumulations of membrane vesicle-like structures and were not fibrillar. Together, these results showed that GPI anchoring directs the assembly of Sup35NM into non-fibrillar, membrane-bound aggregates that resemble PrPSc, raising the possibility that GPI anchor-dependent modulation of protein aggregation might occur with other amyloidogenic proteins. This may contribute to differences in pathogenesis and pathology between prion diseases, which uniquely involve aggregation of a GPI-anchored protein, versus other protein misfolding diseases.

Highly Efficient Prion Transmission by Blood Transfusion

It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 103ID50 as measured by intracerebral inoculation of tg338 mice (ID50 IC in tg338). This was consistent with a whole blood titer greater than 103.6 ID50 IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation.

PrPSc detection in formalin-fixed paraffin-embedded tissue by ELISA

BackgroundFormalin-fixed paraffin-embedded tissue is regularly employed in the diagnosis of transmissible spongiform encephalopathies (TSE) by immunohistochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. While IHC affords advantages over diagnostic approaches that typically utilize fresh or frozen tissue, such as Western blot and ELISA, the process of fixing, staining, and analyzing individual sections by hand does not allow for rapid or high throughput screening. However, preservation of tissues in formalin is not dependent upon the availability of refrigeration.FindingsFormalin-fixed paraffin-embedded tissues from TSE transmission studies of scrapie in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 μm thickness. Samples containing the tissue equivalent of as little as one 5 μm section can be used to readily discriminate positive from negative samples.ConclusionsThis approach cannot replace IHC but may be used along with IHC as both a more rapid and readily high throughput screen where fresh or frozen tissues are not available or impractical.

Fate of Prions in Soil: A Review

Prions are the etiological agents of transmissible spongiform encephalopathies (TSSEs), a class of fatal neurodegenerative diseases affecting humans and other mammals. The pathogenic prion protein is a misfolded form of the host-encoded prion protein and represents the predominant, if not sole, component of the infectious agent. Environmental routes of TSE transmission areimplicated in epizootics of sheep scrapie and chronic wasting disease (CWD) of deer, elk, and moose. Soil represents a plausible environmental reservoir of scrapie and CWD agents, which can persist in the environment for years. Attachment to soil particles likely influences the persistence and infectivity of prions in the environment. Effective methods to inactivate TSE agents in soil are currently lacking, and the effects of natural degradation mechanisms on TSE infectivity are largely unknown. An improved understanding of the processes affecting the mobility, persistence, and bioaviailability of prions in soil is needed for the management of TSE-contaminated environments.

Surveillance for Transmissible Spongiform Encephalopathy in Scavengers of White-Tailed Deer Carcasses in the Chronic Wasting Disease Area of Wisconsin

Chronic wasting disease (CWD), a class of neurodegenerative transmissible spongiform encephalopathies (TSE) occurring in cervids, is found in a number of states and provinces across North America. Misfolded prions, the infectious agents of CWD, are deposited in the environment via carcass remains and excreta, and pose a threat of cross-species transmission. In this study tissues were tested from 812 representative mammalian scavengers, collected in the CWD-affected area of Wisconsin, for TSE infection using the IDEXX HerdChek enzyme-linked immunosorbent assay (ELISA). Only four of the collected mammals tested positive using the ELISA, but these were negative when tested by Western blot. While our sample sizes permitted high probabilities of detecting TSE assuming 1% population prevalence in several common scavengers (93%, 87%, and 87% for raccoons, opossums, and coyotes, respectively), insufficient sample sizes for other species precluded similar conclusions. One cannot rule out successful cross-species TSE transmission to scavengers, but the results suggest that such transmission is not frequent in the CWD-affected area of Wisconsin. The need for further surveillance of scavenger species, especially those known to be susceptible to TSE (e.g., cat, American mink, raccoon), is highlighted in both a field and laboratory setting.

Pathogenetical significance of porencephalic lesions associated with intracerebral inoculation of sheep with the bovine spongiform encephalopathy (BSE) agent

Decreased rates of transmission of transmissible spongiform encephalopathies (TSEs) to sheep have been attributed to some polymorphisms of the prion protein (PrP) and to a 'species barrier' on interspecies experiments. In addition, the blood-brain barrier may be a further impediment to TSE neuroinvasion. The intracerebral (I/C) route is generally considered the most efficient for TSE transmission, as it may help to bypass those factors. Therefore, susceptibility of particular species to specific TSE agents is conducted by this route. This study characterizes the traumatic brain lesions associated with the I/C injection of the bovine spongiform encephalopathy agent in sheep, assesses the relevance of such lesions in the outcome of clinical disease and provides insight into the mechanisms of PrP(d) conversion and amplification following I/C challenge. A total of 27 hemibrains have been macroscopically and immunohistochemically examined to investigate the presence of lesions compatible with the needle track and the PrP(d) distribution, respectively. No residual inoculum was found and the extension and severity of the traumatic brain lesions were unrelated to the clinical outcome. Sheep with PrP(d) accumulation in the brain also showed conspicuous focal aggregates in the porencephalic lesions and in the circumventricular organs. In contrast, sheep without PrP(d) deposits in the brain were also negative in the traumatic lesions. Overall, these findings suggest that the efficiency of the I/C route is due to effective absorption and blood recirculation of infection, rather than to primary amplification at the site of injection.

Prions in Milk from Ewes Incubating Natural Scrapie

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.

Excretion of Transmissible Spongiform Encephalopathy Infectivity in Urine

The route of transmission of most naturally acquired transmissible spongiform encephalopathy (TSE) infections remains speculative. To investigate urine as a potential source of TSE exposure, we used a sensitive method for detection and quantitation of TSE infectivity. Pooled urine collected from 22 hamsters showing clinical signs of 263K scrapie contained 3.8 +/- 0.9 infectious doses/mL of infectivity. Titration of homogenates of kidneys and urinary bladders from the same animals gave concentrations 20,000-fold greater. Histologic and immunohistochemical examination of these same tissues showed no indications of inflammatory or other pathologic changes except for occasional deposits of disease-associated prion protein in kidneys. Although the source of TSE infectivity in urine remains unresolved, these results establish that TSE infectivity is excreted in urine and may thereby play a role in the horizontal transmission of natural TSEs. The results also indicate potential risk for TSE transmission from human urine-derived hormones and other medicines.

Blood clearance of the prion protein introduced by intravenous route in sheep is influenced by host genetic and physiopathologic factors

The risk of transmissible spongiform encephalopathy (TSE) transmission by blood transfusion is dependent on the blood concentrations of the pathologic isoform of prion protein (PrPsc) but may also be influenced by blood concentrations of cellular PrP (PrPc). These concentrations are controlled by the blood clearance of PrP, which has never been evaluated. The blood (actually plasma) clearance of ovine purified prokaryote recombinant PrP (rPrP) was measured in genotyped and in nephrectomized sheep. The exposure to proteinase K-resistant fragments of PrP (PrPres) after intravenous (IV) administration of scrapie-associated fibrils (SAFs) was also investigated in a sheep. The ARR variant of rPrP was eliminated more rapidly than its VRQ counterpart. The PrPc plasma concentrations in homozygous highly susceptible VRQ sheep were greater than in homozygous ARR-resistant sheep, suggesting that clearance of the ARR variant of PrPc was higher than that of the VRQ variant. The plasma clearance of rPrP was decreased by 52 percent after a bilateral nephrectomy indicating the significant contribution of the kidneys in eliminating rPrP. PrPres was shown to be slowly eliminated after IV administration of scrapie-associated fibrils. PrP host genotype and physiopathologic factors could influence the risk of TSE transmission by modulating blood PrP clearance. This risk was increased by the sustained exposure to PrPres after IV administration. It should be noted that although the materials that have been administered (rPrP and SAFs) were not the actual species of interest, they can be of value as probes for investigating PrP clearance mechanisms.

Safety procedures of coagulation factors

Two main types of safety procedures must be applied to biological products, including plasma derivatives: (i) preventive procedures and (ii) elimination procedures.Prevention includes epidemiological control of donor populations; checks on each donor’s health condition; analysis of each donation for the main pathogens using serological methods; additional analysis of all plasma for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus (HAV) and the B19 virus, using nucleic acid amplification techniques (NAT). A 60 days or longer inventory hold of all plasma donations is applied, to allow additional time to discard previous donations from potential seroconverting or otherwise rejectable donors.Elimination procedures minimize the low residual risk of transmitting pathogens, including unknown or previously undetected ones. Since the introduction 20 years ago of solvent‐detergent treatment, very effective against enveloped viruses (HIV, HBV, HCV, West Nile virus, SARS, avian influenza virus etc), there have been no known cases of transmission of this type of pathogens by products manufactured according to this procedure. Other inactivation procedures such as pasteurization, dry‐heat or nanofiltration may prove equally effective. In addition, dry‐heat treatment and nanofiltration are capable of effectively eliminating non‐enveloped viruses (HAV, B19 virus). Recent studies show that the B19 virus is much more sensitive to heat (in lyophilized state or by pasteurization) and acid pH than previously thought.Although there is no evidence for the transmission of classic transmissible spongiform encephalopathies (TSEs) through blood or blood‐products transfusion, four possible cases have been reported in the United Kingdom involving transmission by non‐leukoreduced blood components of the agent that causes variant Creutzfeldt‐Jakob Disease (vCJD), a disease linked to the outbreak of bovine spongiform encephalopathy (BSE) which took place in that country. However, there are no cases of human TSE (classic or variant) transmission by plasma‐derived products. Analytical methods capable of detecting the vCJD agent in patients’ brains (where high titres are found) and other tissues (such as the spleen, appendix and lymph nodes, where much lower concentrations are found) are unable to detect the agent in blood or plasma from patients with vCJD, even in the clinical phase of the disease. Experiments by Grifols and other groups show that the capacity of the production processes to eliminate vCJD agent models is many orders of magnitude greater than the maximum expected load of the agent. In this regard, the efficacy of precipitation, affinity chromatography, depth filtration and nanofiltration are particularly notable.

Safe Handling of Transmissible Spongiform Encephalopathies Specimens in the Histopathology Laboratory

Creutzfeldt-Jakob disease (CJD) is the most common form of the human transmissible spongiform encephalopathies (TSEs), a term that describes a fatal degenerative condition of the central nervous system, and has a worldwide death rate of about 1 case per million people per year. Approximately 90% of CJD cases are classified as sporadic, whereas approximately 10% are familial. It is the <1% of CJD cases that result from iatrogenic exposure from direct inoculation, implantation, or transplantation of infectious material that concern laboratory personnel. A consistent theory of TSEs is that a normal neuronal membrane protein-prion protein encoded by a gene on chromosome 20 and composed solely of protein becomes altered to a protease-resistant form accumulates in brain and the central nervous system and is exhibited by the replacement of normal neurons (photo A), with microscopic vacuoles (photo B) the feature from which TSE derives its name. Prions have exceptional resistance to chemical and physical decontamination methods. The infective agent is not inactivated by tissue fixatives or common disinfectants, and some infectivity may survive standard laboratory autoclaving methods (e.g., 121°C for 15 min). Therefore, it is prudent for the histopathology laboratory to take a precautionary approach for safe handling and processing of brain specimens from known or suspected cases of CJD to protect against possible TSE transmission to laboratory personnel (The J Histotechnol 30:81, 2007).Submitted February 15, 2007; accepted with revisions March 20, 2007.

Halting the spread of human prion disease – exceptional measures for an exceptional problem

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are uniformly fatal neurodegenerative diseases and include Transmissible Mink Encephalopathy in mink, scrapie in sheep, Bovine Spongiform Encephalopathy (BSE) in cattle and large cats, Chronic Wasting Disease (CWD) in deer and Creutzfeldt Jakob disease (CJD) in humans. They are characterised by a long incubation period, normally several months or years, but without evidence of an infecting agent or an immune response to one.1 The exact mode of transmission of TSEs is a matter of debate, but it is known that a host protein, the prion protein (PrP), plays a central role in these diseases. The prion protein adopts two main conformations: a normal, cellular form (PrPc) expressed in most cells, and a disease-associated form (PrPsc), usually found in brain, spleen and lymph node follicular dendritic cells, especially in tonsils. The altered structure of PrPsc is characterised by its lower alpha-helical and higher beta-sheet content, and by its partial resistance to proteinase K digestion. It is thought that PrPsc replicates by acting as a conformational template, causing the host PrPc to adopt the PrPsc conformation. As disease develops, large protein fibrils (called scrapieassociated fibrils, which contain aggregated PrPsc molecules) are seen in neuronal and lymphoid tissues.2 In the absence of a unique nucleic acid or an immune response, detection of the agent and diagnosis of the disease is difficult. Moreover, it has been known for some time that some TSE strains are resistant to steam sterilisation and most other common disinfection procedures. Consequently, a number of cases of iatrogenic CJD have been attributed to the failure of cleaning and sterilising procedures to effectively decontaminate medical and surgical instruments.3 The BSE agent is especially resistant to steam sterilisation and recent work has shown that the variant CJD (vCJD) agent is similarly resistant, with the standard autoclave cycle currently in use in the UK’s national health Service (NHS) reducing titres in infected brain with an estimated titre of 109 infectious units/gram by only 2 or 3 orders of magnitude. As the disease-forming characteristics of the agent rely on its secondary structure and not on the integrity of a nucleic acid sequence, many sterilisation processes such as UV irradiation are ineffective. Moreover, other inactivation processes such as fixation with formalin and other aldehydes preserve the infectious nature of the agent as they cross-link proteins and thus lock in their secondary structure. This is unlike conventional infectious agents such as viruses which require structural fluidity in their proteins to enter the host and replicate. Furthermore, inadequate heating may accelerate dehydration, forming a protective layer which inhibits further inactivation.

Ser170 controls the conformational multiplicity of the loop 166–175 in prion proteins: implication for conversion and species barrier

The self-perpetuating conversion of cellular prion proteins (PrP(C)) into an aggregated beta-sheet rich conformation is associated with transmissible spongiform encephalopathies (TSE). The loop 166-175 (L1) in PrP(C), which displays sequence and structural variation among species, has been suggested to play a role in species barrier, in particular against transmission of TSE from cervids to domestic and laboratory animals. L1 is ordered in elk PrP, as well as in a mouse/elk hybrid (in which L1 of mouse is replaced by elk) but not in other species such as mice, humans, and bovine. To investigate the source and significance of L1 dynamics, we carried out explicit solvent molecular dynamics simulations (approximately 0.5 micros in total) of the mouse prion protein, the mouse/elk hybrid, and control simulations, in which the mouse sequence is reintroduced into the structure of the mouse/elk hybrid. We found that the flexibility of L1 correlates with the backbone dynamics of Ser170. Furthermore, L1 mobility promotes a substantial displacement of Tyr169, rupture of the Asp178-Tyr128 and Asp178-Tyr169 side chain hydrogen bonds, as well as disruption of Tyr169-Phe175 pi-stacking interaction. The simulation results go beyond the available experimental data because they highlight the dependence of this network of interactions on residue 170 and L1 plasticity.

Prion Clearance Capacity Is Improved by Modification of a Filtration Step in the Gamunex® (IGIV) Manufacturing Process.

Prions are misfolded pathogenic proteins associated with the transmission of transmissible spongiform encephalopathies (TSEs). Although no TSE transmission through human plasma products has been documented to date, the risk of transmission exists. To provide assurance for product safety with respect to the risk of TSE transmission, this study evaluates the prion clearance potential of a modified intravenous immunoglobulin (Gamunex®) manufacturing process (Figure 1). [Display omitted] In the process prior to modification, an intermediate material was subjected to cloth filtration. In the modified process, the cloth filtration is replaced with a depth filtration to improve pathogen removal capacity. Both processes were studied using validated scaled-down models, along with a spiked prion agent. The modified process reduced the prion level to below the limit of detection by Western blot assay. The animal bioassay for infectivity confirmed that the modified process has a marked improvement in prion clearance. This modified process has been implemented in actual Gamunex® manufacturing process. Gamunex® is the only liquid IGIV with FDA approved prion clearance data.

CJD PrP sc removal by nanofiltration process: Application to a therapeutic immunoglobulin solution (Lymphoglobuline ®)

The characteristic of transmissible spongiform encephalopathies (TSE) is an accumulation of partially protease resistant (PrP res) abnormal prion protein (PrP sc). This pathological prion protein is very resistant to conventional inactivation methods. The risk of transmission of TSE, such as Creutzfeldt–Jakob disease (CJD), by biopharmaceutical products prepared from human cells must be taken into account. The nanofiltration process has been proved to be effective in removing viruses and scrapie agent. The major advantages of this technique are flexibility and efficacy in removing infectious particles without altering biopharmaceutical characteristics and properties. This study focused on the removal of human PrP sc by means of a nanofiltration method after spiking a Lymphoglobuline ® solution with a CJD brain homogenate. Lymphoglobuline ® equine anti-human thymocyte immunoglobulin is a selective immunosuppressive agent acting mainly on human T lymphocytes. The therapeutic indications are: • immunosuppression for transplantation: prevention and treatment of graft rejection; • treatment of aplastic anemia. In our study, CJD homogenate was spiked at three different dilutions (low, moderate and high) in the Lymphoglobuline ® product. The nanofiltration process was performed on each sample. Using the western blot technique, the PrP res signal detected in nanofiltrates was compared to that obtained with a reference scale (dilution series of CJD brain homogenate in Lymphoglobuline ® detected by western blot and elaborated on 3.3 log). After nanofiltration, the PrP res western blot signal was detected with a significant reduction in the less dilute sample, whereas the signal was undetectable in the two other samples. These are the first data in CJD demonstrating a clearance between 1.6 and 3.3 log with a Lymphoglobuline ® recovery of over 93%. The nanofiltration process confirms its relative efficacy in removing human CJD PrP sc.

Clearance of Prions During Plasma Protein Manufacture

Protein products isolated from human plasma are an important class of therapeutics that are used to treat patients afflicted with hereditary deficiencies, trauma, and severe infections. Because of the human origin of the starting material for the production of these biological products, there is a risk of transmitting infectious agents, including viruses and the infectious agents that cause transmissible spongiform encephalopathies (TSEs). The agent that is thought to cause TSEs is a disease-associated, misfolded form of the prion protein or PrP(Sc). Unlike viruses, there are no donor screening tests for TSEs available, and PrP(Sc) is resistant to traditional viral inactivation methods. Therefore, manufacturers of plasma products are faced with special challenges to ensure product safety with respect to TSEs. Fortunately, a growing body of evidence supports the capacity of manufacturing processes to remove infectious prions from the product stream during the purification of plasma products. This can be attributed in part to the unusual physicochemical nature of PrP(Sc), which is distinct from that of soluble therapeutic proteins. Although there is no reported TSE transmission through the use of plasma products to date, many unknowns remain to be addressed through long-term epidemiologic monitoring and further experimental studies.

Solvent-Detergent Treatment and Ion-Exchange Chromatography Have No Impact on Prion Removal by 15 nm Nanofiltration in the Manufacture of Factane®.

The transmission risk for new variant Creutzfeldt-Jakob Disease by medicinal plasma-derived products (MPP) is considered as very low, even theoretical, as to date no cases associated with these products have been reported. As a precautionary measure, European Regulatory authorities require that manufacturers of MPP perform an assessment of their manufacturing processes capacity to remove transmissible spongiform encephalopathy (TSE) agents. In the absence of scientific data, manufacturers are required to perform specific studies demonstrating their processes efficacy towards prion removal. We investigated the efficacy of 3 manufacturing steps in the Factane® process: step 1: precipitation combined with alumina gel adsorption and filtration through positively charged membranes; step 2: ion-exchange chromatography in the presence of Solvent-Detergent (SD) and step 3: combined 35 nm + 15 nm nanofiltration. Removal of TSE infectivity was assessed either by independent experiments, to determine the reduction factor associated with each individual step, or by a study combining several steps in order to investigate the impact of upstream process steps (SD treatment and Chromatography) on the efficacy of nanofiltration. Intermediate products collected from industrial batches were spiked with a microsomal fraction purified from a hamster brain homogenate (scrapie 263K strain). Detection and titration of the infectious agent were performed by Western Blot (WB) and/or Bioassay. When each step was studied individually, step 1 led to a reduction factor of 1.5 log10 (WB, clearance factor: 3 log10) and step 2 to a reduction factor of 1.7 log10 (WB). Step 3 led to reduction factors higher than or equal to 3.3 log10 by WB as well as by Bioassay. In a study combining steps 2 and 3, the total reduction factor was higher than or equal to 5.1 log10 (WB). The bioassay corresponding to this combined study is ongoing. Results from investigational studies of the Factane® manufacturing process highlight the contribution of the 3 identified steps towards prion removal which is in line with previously reported data. Nanofiltration shows a very significant contribution to the removal of TSE agents as evidenced by WB and bioassay experiments. This observation is in agreement with the estimated size of TSE agents (15-45 nm) as well as with their biophysical properties (agregability, insolubility and hydrophobicity). Data from the combined study (reduction factor ≥ 5.1 log10) are compatible with the cumulative effects of the chromatography and the nanofiltration steps. This observation shows that nanofiltration's efficiency in removing prions is unchanged by the upstream treatments, namely SD treatment, and supports the hypothesis that the overal global reduction factor for the manufacture process is equal to the sum of the individual steps. These data confirm the interest of nanofiltration as an additional safety step in increasing the safety margin of plasma products towards the theoretical TSE transmission risk.

Prion biology relevant to bovine spongiform encephalopathy1

Bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) of deer and elk are a threat to agriculture and natural resources, as well as a human health concern. Both diseases are transmissible spongiform encephalopathies (TSE), or prion diseases, caused by autocatalytic conversion of endogenously encoded prion protein (PrP) to an abnormal, neurotoxic conformation designated PrPsc. Most mammalian species are susceptible to TSE, which, despite a range of species-linked names, is caused by a single highly conserved protein, with no apparent normal function. In the simplest sense, TSE transmission can occur because PrPsc is resistant to both endogenous and environmental proteinases, although many details remain unclear. Questions about the transmission of TSE are central to practical issues such as livestock testing, access to international livestock markets, and wildlife management strategies, as well as intangible issues such as consumer confidence in the safety of the meat supply. The majority of BSE cases seem to have been transmitted by feed containing meat and bone meal from infected animals. In the United Kingdom, there was a dramatic decrease in BSE cases after neural tissue and, later, all ruminant tissues were banned from ruminant feed. However, probably because of heightened awareness and widespread testing, there is growing evidence that new variants of BSE are arising "spontaneously," suggesting ongoing surveillance will continue to find infected animals. Interspecies transmission is inefficient and depends on exposure, sequence homology, TSE donor strain, genetic polymorphism of the host, and architecture of the visceral nerves if exposure is by an oral route. Considering the low probability of interspecies transmission, the low efficiency of oral transmission, and the low prion levels in nonnervous tissues, consumption of conventional animal products represents minimal risk. However, detection of rare events is challenging, and TSE literature is characterized by subsequently unsupported claims of species barriers or absolute tissue safety. This review presents an overview of TSE and summarizes recent research on pathogenesis and transmission.

Follicular dendritic cell dedifferentiation reduces scrapie susceptibility following inoculation via the skin

Transmissible spongiform encephalopathies (TSEs) are a group of subacute infectious neurodegenerative diseases that are characterized by the accumulation in affected tissues of PrP(Sc), an abnormal isoform of the host prion protein (PrPc). Following peripheral exposure, TSE infectivity and PrP(Sc) usually accumulate in lymphoid tissues prior to neuroinvasion. Studies in mice have shown that exposure through scarified skin is an effective means of TSE transmission. Following inoculation via the skin, a functional immune system is critical for the transmission of TSEs to the brain, but until now, it has not been known which components of the immune system are required for efficient neuroinvasion. Temporary dedifferentiation of follicular dendritic cells (FDCs) by treatment with an inhibitor of the lymphotoxin-beta receptor signalling pathway (LTbetaR-Ig) 3 days before or 14 days after inoculation via the skin, blocked the early accumulation of PrP(Sc) and TSE infectivity within the draining lymph node. Furthermore, in the temporary absence of FDCs before inoculation, disease susceptibility was reduced and survival time significantly extended. Treatment with LTbetaR-Ig 14 days after TSE inoculation also significantly extended the disease incubation period. However, treatment 42 days after inoculation did not affect disease susceptibility or survival time, suggesting that the infection may have already have spread to the nervous system. Together these data show that FDCs are essential for the accumulation of PrP(Sc) and infectivity within lymphoid tissues and subsequent neuroinvasion following TSE exposure via the skin.