Role of donor genotype in RT-QuIC seeding activity of chronic wasting disease prions using human and bank vole substrates.

Chronic wasting disease is a transmissible spongiform encephalopathy of cervids. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein (PrPC) to pathogenic conformers (PrPSc), and the pathogenic forms accumulate in the brain and other tissues. Real-time Quaking Induced Conversion (RT-QuIC) can be used for the detection of prions and for prion strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how either PrPSc from cervids of different genotypes or PrPSc from different sources of CWD influence the fibril formation of recombinant bank vole (BV) or human prion proteins using RT-QuIC. We found that reaction mixtures seeded with PrPSc from different genotypes of white-tailed deer or reindeer brains have similar conversion efficiency with both substrates. Also, we observed similar results when assays were seeded with different sources of CWD. Thus, we conclude that the genotypes of all sources of CWD used in this study do not influence the level of conversion of PrPC to PrPSc.

Analysis of the Glycosylation Profile of Disease-Associated Water-Soluble Prion Protein Using Lectins.

The disease-associated water-soluble form of hamster prion protein (ws-PrPSc) has recently been found to be less stable than classical PrPSc. Since the stability of PrP to degradation correlates with its glycosylation level, the aim of this study was to investigate whether there are differences between the glycosylation of ws-PrPSc and classical PrPSc of hamster which might account for the ws-PrPSc minor stability compared with that of the classical PrPSc. Thus, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (SHS) and high-speed pellet (PHS)] and blood plasma by anti-PrP antibodies (3F4 and 6H4) and subjected to screening for glycans by lectins under denaturing or nondenaturing procedures in a sandwich lectin-ELISA. Glycans have been found in minor quantities and differently exposed on ws-PrPSc from SHS and plasma compared with classical PrPSc from PHS. These differences have been shown to be potentially responsible for the instability of ws-PrPSc. Treatment of infected blood with GdnHCl significantly (P<0.01) increased the detection of ws-PrPSc in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrPSc stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrPSc glycosylation with particular regard to the mannose sugar. Analysis of lectins immunoreactivity toward total proteins from plasma collected before and at different time points after infection revealed that mannose might exert a stabilizing effect toward all of hamster blood glycoproteins, regardless of scrapie infection. Since low levels of ws-PrPSc/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrPC to convert to ws-PrPSc both in blood and the brain. Therefore, PrPC glycosylation characteristics may provide a tool for the determination risk of prion transmissibility.

A novel approach for scrapie-associated prion (PrPSc) detection in blood using the competitive affinity of an aggregate-specific antibody and streptavidin to PrPSc.

Scrapie is a fatal neurodegenerative disorder affecting sheep and goats, originating from exposure to disease-associated prions (PrPSc). An ante-mortem screening test that can detect native PrPSc in body fluids remains unavailable due to insufficient sensitivity of current detection methods that involve proteinase or denaturation treatments. We adopted an approach to detect PrPSc in whole blood using a simple proteinase- and denaturation-independent immunoassay, based on the competitive affinity of an aggregate-specific monoclonal antibody and streptavidin to PrPSc. First, we demonstrated the ability of native PrPSc to bind to streptavidin and the inhibition of this interaction by 15B3 antibody (P<0.05). This led to a new two-step assay that involved capturing native prions from infected blood on a solid-state matrix and detection of PrPSc aggregates by evaluating the conformation-dependent conjugate catalytic activity ratio in samples against a pre-determined threshold. This test showed capacity for detecting scrapie prions in 500µl of sheep whole blood spiked with scrapie brain homogenate containing approximately 5ng of total brain protein, and estimated to have 500fg of PrPSc. The test also discriminated between blood samples from scrapie-negative (6 sheep, 4 goats) and scrapie-infected animals (3 experimentally infected sheep, 7 naturally infected goats). Collectively, with the proposed high-throughput sample-processing platform, these initial studies provide insights into the development of a large-scale screening test for the routine diagnosis of scrapie.

The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases.

The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrPSc) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrPSc seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays-such as protein misfolding cyclic amplification (PMCA)-which are based on sonication, the RT-QuIC technique is based on prion seed-induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes ∼87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimer's and Parkinson's disease.

Ultrasensitive detection of PrP(Sc) in the cerebrospinal fluid and blood of macaques infected with bovine spongiform encephalopathy prion.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrP(Sc)) in the central nervous system. The pathological features and biochemical properties of PrP(Sc) in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt-Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrP(Sc). This method involves amplifying PrP(Sc) by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrP(C) substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrP(Sc) in the CSF by serial PMCA, and the CSF levels of PrP(Sc) tended to increase with disease progression. In addition, PrP(Sc) was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrP(Sc) in non-human primate models of CJD.

Investigation of serum protein profiles in scrapie infected sheep by means of SELDI-TOF-MS and multivariate data analysis.

BACKGROUND: Classical scrapie in sheep is a fatal neurodegenerative disease associated with the conversion PrPC to PrPSc. Much is known about genetic susceptibility, uptake and dissemination of PrPSc in the body, but many aspects of prion diseases are still unknown. Different proteomic techniques have been used during the last decade to investigate differences in protein profiles between affected animals and healthy controls. We have investigated the protein profiles in serum of sheep with scrapie and healthy controls by SELDI-TOF-MS and LC-MS/MS. Latent Variable methods such as Principal Component Analysis, Partial Least Squares-Discriminant Analysis and Target Projection methods were used to describe the MS data. RESULTS: The serum proteomic profiles showed variable differences between the groups both throughout the incubation period and at the clinical end stage of scrapie. At the end stage, the target projection model separated the two groups with a sensitivity of 97.8%, and serum amyloid A was identified as one of the protein peaks that differed significantly between the groups. CONCLUSIONS: At the clinical end stage of classical scrapie, ten SELDI peaks significantly discriminated the scrapie group from the healthy controls. During the non-clinical incubation period, individual SELDI peaks were differently expressed between the groups at different time points. Investigations of differences in -omic profiles can contribute to new insights into the underlying disease processes and pathways, and advance our understanding of prion diseases, but comparison and validation across laboratories is difficult and challenging.

An overview of the diagnostic tools.

Prions are unconventional infectious agents that cause fatal neurological illnesses such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy, and scrapie. Variant CJD can occur via blood transfusions. However, as no screening assay is available, uncertainties remain over the prevalence of vCJD in asymptomatic blood donors. Development of a diagnostic assay is therefore a primary objective. Little is known about the nature, distribution and level of infectivity in human blood and we have to rely on assumptions made from animal models. Ideally, two types of assays are required: a rapid high-throughput assay to routinely screen all blood donations and a confirmatory assay to ensure that all positive results from initial screening are true positives. Key event in prion disease is thought to be the conversion of normal cellular prion protein PrPc to a misfolded aggregated form termed PrP(TSE). This specific characteristic has been exploited to develop some tests.

Analysis of prion protein aggregates in blood and brain from pre-clinical and clinical BSE cases.

Prion diseases are infectious neurodegenerative diseases affecting humans and animals. The food-borne bovine spongiform encephalopathy (BSE) had serious impact on both economy and public health, respectively. To follow the pathogenesis of BSE, oral challenge studies were previously conducted, among others on the Isle of Riems, Germany (Balkema-Buschmann et al., 2011b). In the present work brain and plasma samples from this pathogenesis study were subjected to surface fluorescence distribution analysis (sFIDA). sFIDA is a diagnostic tool that exploits the aggregated state of the disease-related prion protein (PrP) as a biomarker for prion disorders. With the exception of one animal, all tested brain samples from clinical cattle exhibited a high titer of PrP particles. Moreover we could detect PrP aggregates already 16 and 24 months after infection. In contrast to our previous demonstration of PrP particles in blood plasma from scrapie sheep, however, no aggregates could be identified in plasma from pre-clinical and clinical cattle. This is in accordance with other studies suggesting a restriction of the BSE infection to the central nervous system.

Removal of TSE agent from plasma products manufactured in the United Kingdom.

BACKGROUND AND OBJECTIVES: The outbreak of vCJD in the UK leads to concern regarding the potential for human-to-human transmission of this agent. Plasma-derived products such as albumin, immunoglobulin and coagulation factors were manufactured by BPL from UK plasma up until 1999 when a switch to US plasma was made. In the current study, the capacity of various manufacturing processes that were in use both prior to and after this time to remove the TSE agent was tested. MATERIALS AND METHODS: Small-scale models of the various product manufacturing steps were developed. Intermediates were spiked with scrapie brain extract and then further processed. Samples were assayed for the abnormal form of prion protein (PrP(SC) ) by Western blotting, and the reduction in the amount of scrapie agent determined. RESULTS: Many of the manufacturing process steps produced significant reduction in the scrapie agent. Particularly effective were steps such as ethanol fractionation, depth filtration, ion-exchange and copper chelate affinity chromatography. Virus retentive filters, of nominal pore size 15 or 20 nm, removed >3 log. The total cumulative reduction capacity for individual products was estimated to range from 7 to 14 log. In the case of factor VIII (8Y), the total removal was limited to 3 log. CONCLUSION: All the processes showed a substantial capacity to remove the TSE agent. However, this was more limited for the intermediate purity factor VIII 8Y which included fewer manufacturing steps.

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 10³·6ID50 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.

Initial fate of prions upon peripheral infection: half-life, distribution, clearance, and tissue uptake.

Prion diseases are infectious neurodegenerative disorders associated with the misfolded prion protein (PrP(Sc)), which appears to be the sole component of the infectious agent (termed prion). To produce disease, prions have to be absorbed into the body and reach sufficient quantities in the brain. Very little is known about the biological mechanisms controlling the initial fate of prions. Here, we studied the systemic pharmacokinetics and biodistribution of PrP(Sc) in vivo. After an intravenous injection of highly purified radiolabeled or native unlabeled PrP(Sc), the protein was eliminated rapidly from the serum (half-life of 3.24 h), mostly through tissue uptake. The quantity of intact PrP(Sc) reaching the brain was ∼ 0.2% of the injected dose per gram of brain tissue (ID/g). The highest levels were found in liver (∼ 20% ID/g), spleen (∼ 13% ID/g), and kidney (∼ 7.4% ID/g). Cell surface PrP(C) does not appear to play a role in PrP(Sc) pharmacokinetics, since the infectious protein distributed similarly in wild-type and PrP-null mice. To measure tissue uptake kinetics and biodistribution accurately, vascular space in tissues was measured with radioactively labeled albumin coinjected with radioactively labeled PrP(Sc). Our results provide a fundamental pharmacokinetic characterization of PrP(Sc) in vivo, which may be relevant to estimate tissue risks and mechanisms of prion neuroinvasion and to identify novel therapeutic strategies.

PrP(Sc) is associated with B cells in the blood of scrapie-infected sheep.

Recently, we reported that PrP(Sc), a surrogate marker for prion disease, is associated with the cellular fraction of blood from scrapie-infected sheep using a ligand-based immunoassay. In the study reported here, we found that a subset of peripheral blood mononuclear cells is most likely to sequester PrP(Sc) during both the preclinical phase of disease and at clinical end point. These cells had a cell surface phenotype of MHC class II DQ(+), surface immunoglobulin(+), CD11b(+), CD11c(+), CD21(+/)(-), which is consistent with a subpopulation of B cells. What role these cells play in the pathogenesis of scrapie is unclear, but they may contribute to the trafficking of prions to the spleen during early pathogenesis of the disease. Furthermore, tests for preclinical diagnostics could be further improved by targeting these cells.

Estimating prion concentration in fluids and tissues by quantitative PMCA.

Prions, the proteinaceous infectious agent responsible for prion diseases, can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA) technology. Here we describe a quantitative PMCA procedure to calculate the concentration of very low levels of prions in biological samples. Using this procedure, we determined the quantities of misfolded prion protein (PrP(Sc)) in brain, spleen, blood and urine of scrapie-affected hamsters.

A novel method for preclinical detection of PrPSc in blood.

In this study, we demonstrate that a moderate amount of protein misfolding cyclic amplification (PMCA) coupled to a novel surround optical fibre immunoassay (SOFIA) detection scheme can be used to detect the disease-associated form of the prion protein (PrP(Sc)) in protease-untreated plasma from preclinical and clinical scrapie sheep, and white-tailed deer with chronic wasting disease, following natural and experimental infection. PrP(Sc), resulting from a conformational change of the normal (cellular) form of prion protein (PrP(C)), is considered central to neuropathogenesis and serves as the only reliable molecular marker for prion disease diagnosis. While the highest levels of PrP(Sc) are present in the central nervous system, the development of a reasonable diagnostic assay requires the use of body fluids that characteristically contain exceedingly low levels of PrP(Sc). PrP(Sc) has been detected in the blood of sick animals by means of PMCA technology. However, repeated cycling over several days, which is necessary for PMCA of blood material, has been reported to result in decreased specificity (false positives). To generate an assay for PrP(Sc) in blood that is both highly sensitive and specific, we have utilized limited serial PMCA (sPMCA) with SOFIA. We did not find any enhancement of sPMCA with the addition of polyadenylic acid nor was it necessary to match the genotypes of the PrP(C) and PrP(Sc) sources for efficient amplification.

Anti-PrP antibodies detected at terminal stage of prion-affected mouse.

The causative agent of prion diseases is the pathological isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc has an identical amino acid sequence to PrPC; thus, it has been assumed that an immune response against PrPSc could not be found in prion-affected animals. In this study, we found the anti-prion protein (PrP) antibody at the terminal stage of mouse scrapie. Several sera from mice in the terminal stage of scrapie reacted to the recombinant mouse PrP (rMPrP) molecules and brain homogenates of mouse prion diseases. These results indicate that mouse could recognize PrPC or PrPSc as antigens by the host immune system. Furthermore, immunization with rMPrP generates high titers of anti-PrP antibodies in wild-type mice. Some anti-PrP antibodies immunized with rMPrP prevent PrPSc replication in vitro. The mouse sera from terminal prion disease have several wide epitopes, although mouse sera immunized with rMPrP possess narrow epitopes.

Discrimination between prion-infected and normal blood samples by protein misfolding cyclic amplification.

BACKGROUND: Diagnosis of prion disease from blood samples requires the detection of minute quantities of misfolded protein (PrP(Sc)) against a high background of correctly folded material (PrP(C)). Protein misfolding cyclic amplification (PMCA) is a technique that can amplify small amounts of seed PrP(Sc) to a level detectable by conventional methods. Application of PMCA to the testing of whole blood samples enhances the ability to detect PrP(Sc) and allows antemortem detection of prion infection and could facilitate blood screening. STUDY DESIGN AND METHODS: The PMCA method was used to detect prion infection in blood samples obtained from mice experimentally infected with prion disease. Mice were culled at various time points throughout the incubation period for disease and subjected to serial PMCA (sPMCA). Amplified samples were then analyzed by Western blotting to confirm the presence or absence of infection. RESULTS: After sPMCA, blood samples from Rocky Mountain Laboratory-infected mice showed amplification of PrP(Sc) to levels readily detectable by Western blotting. Control samples obtained from mice mock inoculated with sterile phosphate-buffered saline did not yield any amplification products. CONCLUSION: sPMCA performed on small volumes of whole blood gave amplification of PK-resistant material to a level detectable by standard methods. Discrimination between infected and control samples was achieved without the need for processing or fractionation of whole blood. The use of whole blood as an analyte circumvents the need to identify the optimal blood compartment for analysis and guarantees the totality of misfolded PrP will be available for detection.

Differentiating blood samples from scrapie infected and non-infected hamsters by detecting disease-associated prion proteins using Multimer Detection System.

This communication describes the application of a modified sandwich enzyme-linked immunosorbent assay (ELISA), termed Multimer Detection System (MDS) for the detection of disease-associated multimeric forms of the prion protein (PrPd) in hamster blood. PrPd was detected in plasma of prion-affected hamsters while MDS revealed no PrPd in identically-treated plasma of healthy animals. This is the first report of a single ELISA- based immune detection of PrPd from blood samples.

Evaluation of removal of prion infectivity from red blood cells with prion reduction filters using a new rapid and highly sensitive cell culture-based infectivity assay.

BACKGROUND: The clearance of infectious prions from biologic fluids is usually quantified by bioassays based on intracerebral inoculation of hamsters or mice; these tests are slow, cumbersome, imprecise, and very expensive. In the present study we describe the use of a new and highly sensitive cell culture-based infectivity assay to evaluate the performance of several prion removal prototype filters. STUDY DESIGN AND METHODS: Five units of 1- to 2-day-old ABO-compatible human red blood cells (RBCs) in saline-adenine-glucose-mannitol were obtained from an AABB-accredited blood bank. The 5 units were combined to create a homogenous pool. Scrapie-infected mouse brain homogenate of a Rocky Mountain Laboratory strain was added to the pooled RBCs. The pooled RBCs were divided into 300-mL aliquots, which were filtered with either standard leukoreduction filter or four prototypes of prion reduction filter. The levels of prion infectivity in the pre- and postfiltration samples were measured with a cell culture-based standard scrapie cell assay (SSCA). RESULTS: All the 22-layer prion reduction filters removed prion infectivity below the limit of detection of the SSCA (reduction in prion infectivity > or =2.0 log(10)LD(50)/mL) while the 10-layer variant showed some residual infectivity. CONCLUSIONS: These results demonstrate the utility of a highly sensitive cell culture-based infectivity assay for screening prion reduction filters. The use of this type of in vitro infectivity assay will substantially help expedite the screening and discovery of devices aimed at reducing the risk of variant Creutzfeldt-Jakob disease transmission through blood transfusion.

A rapid method for detection of PrP by surface plasmon resonance (SPR).

Surface plasmon resonance was used to develop a rapid, label-free and sensitive immunoassay for detection of Prion protein (PrP). Anti-PrP monoclonal antibodies immobilized on the biosensor surface were allowed to bind various concentrations of cellular prion protein (PrP(C)), followed by a pulse with additional soluble anti-PrP polyclonal antibodies to intensify the signal. The interaction of antibody with antigen was monitored in real time. With this method, it was possible to detect PrP(C) with a limit of 31.7 ng/ml in serum and 13.1 ng/ml in HEPES-saline, requiring 1 h for a single sample measurement. The assay also detected misfolded prion protein (PrP(Sc)) in spiked serum and PrP(Sc) in scrapie-infected mouse brains. This is a rapid and sensitive assay for the detection of PrP in serum that could be developed into a platform for a serum-based TSE test.