Plasminogen-Based Capture Combined with Amplification Technology for the Detection of PrPTSE in the Pre-Clinical Phase of Infection

Background

Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP^TSE) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP^TSE concentrations in the femtomolar range.

Methodology/Principal Findings

We have developed a three-step assay that firstly captures PrP^TSE from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP^TSE detection by western blot. We achieved a PrP^TSE capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP^TSE in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP^TSE in human plasma spiked with a 10⁻⁸ dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP^TSE in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

Conclusion/Significance

We have developed a sensitive and specific amplification assay allowing the detection of PrP^TSE in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP^TSE in blood of patients displaying positivity in large scale screening tests.     

Infrared Microspectroscopy Detects Protein Misfolding Cyclic Amplification (PMCA)-induced Conformational Alterations in Hamster Scrapie Progeny Seeds

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP^C) into Proteinase K-resistant, infectious PrP particles (PrP^TSE), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP^C substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP^C substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.     

Rapid and Highly Sensitive Detection of Variant Creutzfeldt - Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies

The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrP^TSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10⁻⁸ brain dilution) of either human vCJD or ovine scrapie PrP^TSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrP^TSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrP^TSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.     

Re-Assessment of PrPSc Distribution in Sporadic and Variant CJD

Human prion diseases are fatal neurodegenerative disorders associated with an accumulation of PrP^Sc in the central nervous system (CNS). Of the human prion diseases, sporadic Creutzfeldt-Jakob disease (sCJD), which has no known origin, is the most common form while variant CJD (vCJD) is an acquired human prion disease reported to differ from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. Peripheral tissue involvement in prion disease, as judged by PrP^Sc accumulation in the tonsil, spleen, and lymph node has been reported in vCJD as well as several animal models of prion diseases. However, this distribution of PrP^Sc has not been consistently reported for sCJD. We reexamined CNS and non-CNS tissue distribution and levels of PrP^Sc in both sCJD and vCJD. Using a sensitive immunoassay, termed SOFIA, we also assessed PrP^Sc levels in human body fluids from sCJD as well as in vCJD-infected humanized transgenic mice (Tg666). Unexpectedly, the levels of PrP^Sc in non-CNS human tissues (spleens, lymph nodes, tonsils) from both sCJD and vCJD did not differ significantly and, as expected, were several logs lower than in the brain. Using protein misfolding cyclic amplification (PMCA) followed by SOFIA, PrP^Sc was detected in cerebrospinal fluid (CSF), but not in urine or blood, in sCJD patients. In addition, using PMCA and SOFIA, we demonstrated that blood from vCJD-infected Tg666 mice showing clinical disease contained prion disease-associated seeding activity although the data was not statistically significant likely due to the limited number of samples examined. These studies provide a comparison of PrP^Sc in sCJD vs. vCJD as well as analysis of body fluids. Further, these studies also provide circumstantial evidence that in human prion diseases, as in the animal prion diseases, a direct comparison and intraspecies correlation cannot be made between the levels of PrP^Sc and infectivity.     

Proteomic profiling of PrP27‐30‐enriched preparations extracted from the brain of hamsters with experimental scrapie

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation in the CNS of a pathological conformer (PrP^TSE) of the host‐encoded cellular prion protein (PrP^C). PrP^TSE has a central role in the pathogenesis of the disease but other factors are likely involved in the pathological process. In this work we employed a multi‐step proteomic approach for the identification of proteins that co‐purify with the protease‐resistant core of PrP^TSE (PrP27‐30) extracted from brains of hamsters with experimental scrapie. We identified ferritin, calcium/calmodulin‐dependent protein kinase α type II, apolipoprotein E, and tubulin as the major components associated with PrP27‐30 but also trace amounts of actin, cofilin, Hsp90α, the γ subunit of the T‐complex protein 1, glyceraldehyde 3‐phosphate dehydrogenase, histones, and keratins. Whereas some of these proteins (tubulin and ferritin) are known to bind PrP, other proteins (calcium/calmodulin‐dependent protein kinase α type II, Hsp90α) may associate with PrP^TSE fibrils during disease. Apolipoprotein E and actin have been previously observed in association with PrP^TSE, whereas cofilin and actin were shown to form abnormal rods in the brain of patients with Alzheimer disease. The roles of these proteins in the development of brain lesions are still unclear and further work is needed to explain their involvement in the pathogenesis of TSEs.     

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.     

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

The development of variant Creutzfeldt-Jakob disease (vCJD) in three recipients of non-leukoreduced red blood cells from asymptomatic donors who subsequently developed the disease has confirmed existing concerns about the possible spread of transmissible spongiform encephalopathies (TSEs) via blood products. In addition, the presence of disease-associated misfolded prion protein (PrP^TSE), generally associated with infectivity, has been demonstrated in the blood of vCJD patients. However, its origin and distribution in this biological fluid are still unknown. Various studies have identified cellular prion protein (PrP^C) among the protein cargo in human blood-circulating extracellular vesicles released from endothelial cells and platelets, and exosomes isolated from the conditioned media of TSE-infected cells have caused the disease when injected into experimental mice. In this study, we demonstrate the detection of PrP^TSE in extracellular vesicles isolated from plasma samples collected during the preclinical and clinical phases of the disease from mice infected with mouse-adapted vCJD and confirm the presence of the exosomal marker Hsp70 in these preparations.     

The pathological prion protein forms ionic conductance in lipid bilayer

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP^TSE). PrP^TSE pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP^TSE on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP^TSE isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer.     

Direct Detection of Disease Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N Terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.Key Words: BSE, scrapie and vCJD Diagnosis, DELFIA®, novel N terminal antibodies     

In vitro amplification of scrapie and chronic wasting disease PrPres using baculovirus-expressed recombinant PrP as substrate

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrP^Sc. Studies showed that recombinant cellular PrP, PrP^C, expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V₁₃₆L₁₄₁R₁₅₄Q₁₇₁ and A₁₃₆F₁₄₁R₁₅₄Q₁₇₁, and one genotype of white-tailed deer (Q₉₅G₉₆, X₁₃₂,Y₂₁₆) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrP^C shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrP^C and supports amplification of PrP^Sc and PrP^CWD derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrP^Sc or PrP^CWD production. Addition of 2 to 20 μM of manganese chloride (MnCl₂) to unseeded PMCA resulted in conversion of recombinant PrP^C to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.     

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP^C) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP^Sc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP^Sc in prion-infected brain homogenates as an initiating seed to convert PrP^C and trigger the self-propagation of PrP^Sc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP^Sc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP^Sc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP^Sc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.     

Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrP^C) to the infectious scrapie form (PrP^Sc). However, the mechanism that exogenous PrP^Sc infects cells and where pathologic conversion of PrP^C to the PrP^Sc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrP^C to the pathologic PrP^Sc form in N2a cells exposed to strain RML PrP^Sc infected brain homogenates, suggesting that a critical determinant of PrP^C conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP^Sc infects cells by lipid raft dependent, macropinocytosis.     

Temperature influences the interaction of ruminant PrPTSE with soil

Ovine scrapie and cervid chronic wasting disease can be transmitted in the absence of animal-to-animal contact, and environmental reservoirs of infectivity have been implicated in their spread and persistence. Investigating environmental factors that influence the interaction of disease-associated PrP with soils is imperative to understanding what is likely to be the complex role of soil in disease transmission. Here, we describe the effects of soil temperature on the binding/desorption and persistence of both ovine scrapie- and bovine BSE-PrP^TSE. Binding of PrP^TSE to a sandy loam soil at temperatures of 4°C, 8–12°C and 25–30°C demonstrated that an increase in temperature resulted in (1) a decrease in the amount of PrP^TSE recovered after 24 h of interaction with soil, (2) an increase in the amount of N-terminal cleavage of the prion protein over 11 d and (3) a decrease in the persistence of PrP^TSE on soil over an 18 mo period.     

Temperature influences the interaction of ruminant PrPTSE with soil

Ovine scrapie and cervid chronic wasting disease can be transmitted in the absence of animal-to-animal contact, and environmental reservoirs of infectivity have been implicated in their spread and persistence. Investigating environmental factors that influence the interaction of disease-associated PrP with soils is imperative to understanding what is likely to be the complex role of soil in disease transmission. Here, we describe the effects of soil temperature on the binding/desorption and persistence of both ovine scrapie- and bovine BSE-PrP^TSE. Binding of PrP^TSE to a sandy loam soil at temperatures of 4°C, 8–12°C and 25–30°C demonstrated that an increase in temperature resulted in (1) a decrease in the amount of PrP^TSE recovered after 24 h of interaction with soil, (2) an increase in the amount of N-terminal cleavage of the prion protein over 11 d and (3) a decrease in the persistence of PrP^TSE on soil over an 18 mo period.     

Direct Detection of Disease-Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N-terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.     

Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies

The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP^Sc amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP^Sc amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP^Sc amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP^C-to-PrP^Sc conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.     

Enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source

Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrP^C) is required for prion replication, the influence of PrP^C abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrP^C was overexpressed. Tg(MoPrP)4112 mice overexpressing PrP^C supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrP^C-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrP^C is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrP^Sc amplification by using concentrated PrP^C source and expands the use of this methodology.     

Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations

Background

Prion diseases are associated with a conformational switch for PrP from PrP^C to PrP^Sc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.

Scope of review

MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 3₁₀ helix.

Major conclusions

Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP^C to PrP^Sc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP^Sc assemblies.     

Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrP(Sc) in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrP(Sc) in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrP(Sc) in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrP(Sc) was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrP(Sc) to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro.     

Different antigenicities of the N‐terminal region of cellular and scrapie prion proteins

Limited information is available about conformational differences between the abnormal isoform of prion protein (PrP^Sc) and cellular prion protein (PrP^C) under native conditions. To clarify conformational differences between these two isoforms, PrP‐deficient mice were immunized with brain homogenates of normal and scrapie‐infected animals. All mice generated anti‐PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrP^Sc consisting of QGSPGGN (PrP41–47) at the N‐terminus. This study demonstrated a conformational dissimilarity at the N‐terminus between PrP^Sc and PrP^C, a finding that may provide novel information about conformational features of PrP^Sc.