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*

Background: Prions can be transmitted by blood transfusion, but their origin and distribution in blood are unknown. Results: Prions were detected in plasma extracellular vesicles from preclinical and clinically sick mice. Conclusion: Prions associate with blood-circulating extracellular vesicles. Significance: These findings provide information about prion distribution in blood and set the groundwork for novel prion removal and disease diagnosis technologies. 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 (PrPTSE), 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 (PrPC) 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 PrPTSE 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 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.
Human transmissible spongiform encephalopathies (TSEs) 2 are fatal neurodegenerative disorders that can develop as spo-radic, genetic, or infectious diseases. In the United Kingdom and France, which have reported the highest number of variant Creutzfeldt-Jakob disease (vCJD) cases due to exposure to bovine spongiform encephalopathy (BSE)-contaminated products (1), uncertainty exists in regard to the number of infected people (2,3), raising concerns about the safety of blood-and plasma-derived products.
Transmission of vCJD through transfusion of non-leukoreduced red blood cells (RBCs) has been shown in four individuals, three of whom developed clinical disease and one who died from non-TSE-related causes, but in which the presence of disease-associated misfolded prion protein (PrP TSE ) in lymphoreticular tissues was indicative of preclinical or subclinical disease (4). In addition, one hemophilic patient might have been infected through treatment with plasma-derived products (5). Importantly, PrP TSE has been detected in whole blood of vCJD patients (6 -8). Moreover, a number of healthy people have been found to harbor this agent in lymphoreticular tissues (3,9,10); however, it is not known whether PrP TSE circulates in the blood of these individuals and whether they will develop the disease later in life. Recently, infectivity was reported in the plasma of two out of four patients affected by sporadic CJD (sCJD) (11). Although similar levels of PrP TSE have been detected in spleens, tonsils, and lymph nodes of vCJD and sCJD patients by Western blotting (12), the identification of this protein in the blood of individuals afflicted with the latter by various methods, including PrP TSE capture coupled to direct immunodetection of surface-bound material (6), capillary electrophoresis (13), and protein misfolding cyclic amplification (PMCA) coupled to surrounding optical fiber immunoassay (12), has been highly elusive for decades, with only two cases recently reported (8). Nevertheless, extensive epidemiological data continue to provide no evidence of human-to-human transmission of sCJD through blood transfusion (14).
Multiple studies showed that TSE infectivity is present in the blood of experimental rodents, sheep with natural and experimental scrapie and experimental BSE, and deer affected with chronic wasting disease (CWD) (15)(16)(17)(18)(19)(20). PrP TSE has been successfully detected in the following: in whole blood of experimentally infected mice and hamsters, in sheep with natural scrapie and experimental BSE, and in white-tailed deer afflicted with CWD (21)(22)(23); in buffy coats of hamsters and sheep experimentally infected with scrapie (24 -26), as well as in plasma of mice and hamsters with scrapie; and in sheep and white-tailed deer naturally and experimentally infected with scrapie and CWD, respectively (27)(28)(29)(30). However, it is still not fully understood in what blood component TSE infectivity and/or PrP TSE reside and whether and how blood contributes to the spread of the disease from the periphery to the brain.
Exosomes are extracellular membrane vesicles of 40 to 200 nm in diameter, secreted by most cell types upon fusion of multivesicular bodies with the plasma membrane. Originally, they were considered to be a cellular mechanism to eliminate unwanted proteins during reticulocyte maturation (31)(32)(33). Further studies have shown the presence of nucleic acid and protein cargo inside exosomes (34 -36). These extracellular vesicles (EVs) are believed to participate in intercellular communication processes and to execute functions regulating the immune response, angiogenesis, coagulation, inflammation, and programmed cell death (37). Importantly, it has been shown that viruses like HIV-1 can take over the cellular machinery for exosome secretion and use it as another mechanism for viral release (38,39). This "Trojan exosome" hypothesis, posited by Gould et al. (39), is currently being evaluated in protein-misfolding neurodegenerative diseases as a possible mechanism for the spread of misfolded proteins (40 -45). Cellular prion protein (PrP C ) and misfolded PrP TSE have been identified in exosomes from various TSE models (46 -55) and intracerebral inoculation of exosomes obtained from TSE-infected cell cultures has caused clinical disease in mice (47,49). Little is known about the distribution of PrP TSE in blood. PrP C has been detected in exosomes isolated from platelets and annexin V-positive EVs released from apoptotic endothelial cells (48,56). These findings raise the possibility that endothelial and blood cells other than platelets may be capable of releasing PrP C and possibly PrP TSE in association with exosomes and other types of EVs, contributing to the transmission of TSEs through blood-derived products (48, 56 -58). Because of their availability in biological fluids, their stability, and their ability to carry specific cargo, exosomes are ideal targets for detection of biomarkers for the diagnosis of various diseases (34, 59 -61). Detection of PrP TSE in EVs obtained from blood or other body fluids (urine, saliva, nasal secretions, and tears) will enable the design of minimally invasive or noninvasive diagnostic tests for TSEs. In this study, EVs, containing exosomes, were isolated from the conditioned media of cell cultures infected with mouse-adapted vCJD (Mo-vCJD) (16) or Fukuoka-1, a mouseadapted isolate from a Gerstmann-Sträussler-Scheinker dis-ease patient (62,63), and from plasma collected periodically during the preclinical and clinical phases from Mo-vCJD-infected mice. They were used to seed serial automated protein misfolding cyclic amplification (saPMCA) reactions followed by PrP TSE detection by Western blotting.
Our findings represent the first evidence of the presence of PrP TSE in EVs obtained from plasma samples from preclinical and clinically sick mice, and they provide proof-of-concept for the design of a novel microvesicle-based diagnostic test for prion diseases.

EXPERIMENTAL PROCEDURES
Ethics Statement-The Institutional Animal Care and Use Committee of the American National Red Cross reviewed and approved the animal protocols numbered 0807-023 and 1006-045. The American National Red Cross maintains a centralized animal care and use program registered by the United States Food and Drug Administration, ensured with the Office of Laboratory Animal Welfare, and accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. Housing and care of animals are consistent with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and other applicable state and local regulations.
Human blood was collected into citrate/phosphate dextrose upon informed written consent under protocol number 1998-18, approved by the Institutional Review Board of the American National Red Cross.

Animals
Mouse Inoculations-Wild type (WT), C57BL/6 (C57BL), and FVB/NCr (FVB) mice (Charles River Laboratories, Andover, MA) were intracerebrally injected with 30 l of a 10 Ϫ2 diluted or a 10-fold serially diluted (10 Ϫ1 to 10 Ϫ7 ) Mo-vCJDinfected brain homogenate, respectively. Control mice received a similar injection of physiological saline. The inoculum titer was determined by the method of Reed and Muench (64) based on the survival rate of FVB mice inoculated with 10 Ϫ1 to 10 Ϫ8 dilutions of Mo-vCJD. FVB mice in the experimental groups were euthanized when they developed clinical signs of TSE. C57BL mice were euthanized periodically during the incubation period (6,9,12,15, and 20 weeks post inoculation (wpi)) and at clinical onset (ϳ23 wpi). Negative controls were euthanized at the end of the experiment, together with the last mouse from the experimental group. TSE in mice was confirmed by detecting PrP TSE in brain extracts by Western blotting and/or immunohistochemistry as described elsewhere (65).
Blood Collection and Processing-Mouse blood was collected into citrate/phosphate dextrose by cardiac puncture under isoflurane (Patterson Veterinary, Devens, MA) anesthesia. Blood samples were usually processed separately, but on some occasions samples from a few mice were pooled together. Samples were spun at 2,300 ϫ g for 10 min, at room temperature in a FA45-24-11 rotor (Eppendorf, Hauppauge, NY). Plasma was collected, aliquoted, and stored at Ϫ80°C.
Substrate Preparation for saPMCA-Mice were euthanized by CO 2 inhalation and perfused with 40 ml of 5 mmol/liter EDTA in PBS by intra-cardiac injection. Brains were collected and frozen at Ϫ80°C until further processing. Brains were homogenized in appropriate volumes of conversion buffer (1% Triton X-100 in PBS with 1ϫ Complete protease inhibitor mixture (Roche Applied Science)) to prepare a 10% (w/v) WT-mouse brain homogenate (MoBH) that was then split into 1-ml aliquots and stored at Ϫ80°C.

Human Samples
Human blood was separated into RBCs, buffy coat, and platelet-rich plasma by centrifugation at 800 ϫ g for 30 min. Plateletrich plasma was centrifuged at 3,900 ϫ g for 16 min. Plateletpoor plasma was collected, aliquoted, and stored at Ϫ80°C. All centrifugations were performed at room temperature in an A-4-44 rotor (Eppendorf).

Cell Culture
Murine spleen-derived stromal cell culture persistently infected with Mo-vCJD (Mo-vCJD/SP-SC), murine bone marrow-derived stromal cell culture persistently infected with Fukuoka-1 (OF1-BMS), and appropriate uninfected control cell cultures (NB/SP-SC and BMS, respectively) were developed in our laboratory and have been described previously (66,67). Cells were plated in 25-cm 2 flasks with 5 ml of Opti-MEM media (Invitrogen) not supplemented with fetal bovine serum and allowed to grow to confluence at 37°C and 5% CO 2 for 2-3 days for EV preparation.

EV Isolation
EV Isolation from Conditioned Media by ExoQuick TM -TC, Optimized for Culture Media and Urine Samples (System Biosciences, Mountain View, CA)-The medium from each cell culture was collected when cells reached confluence and was centrifuged at 3,000 ϫ g for 15 min to remove cells and cell debris. Supernatants were collected, split in 5-ml aliquots, frozen on dry ice, and stored at Ϫ80°C until further processing. Supernatant aliquots of 5 ml were thawed at room temperature, mixed with 1 ml of ExoQuick TM -TC, and incubated at 4°C overnight. The mixture was centrifuged at 1,500 ϫ g for 30 min, and the EV pellets were collected and stored at Ϫ80°C until analysis. All centrifugations were done at 4°C in an A-4-44 rotor.
EV Isolation from Plasma by ExoQuick TM , Optimized for Serum or Ascites (System Biosciences)-Plasma samples were thawed at 37°C and spun for 15 min at 3,000 ϫ g. The resulting supernatant was mixed with ExoQuick TM (63 l of ExoQuick per 250 l of plasma) and incubated overnight at 4°C. Samples were centrifuged at 1,500 ϫ g for 30 min and EV pellets were collected and stored at Ϫ80°C until analysis. All centrifugations were performed at 4°C in a FA45-24-11 rotor.

Exosome Purification Using a 30% Sucrose Cushion
EV pellets obtained after ExoQuick TM were processed through a 30% sucrose cushion as indicated in the text, following a protocol adapted from Thery et al. (68). The ExoQuick TM pellet was thawed at room temperature, resuspended in 4 ml of PBS, and layered on top of 0.7 ml of 30% sucrose prepared in a Tris/D 2 O solution (30 g of sucrose, 2.4 g of Tris base, D 2 O up to 100 ml). After an initial ultracentrifugation at 110,000 ϫ g for 75 min, three fractions were collected as follows: a top ("top") layer of ϳ0.2 ml, the "middle" layer containing the sucrose layer and the PBS/sucrose interface, and the pellet ("bottom"). The pellet was resuspended in PBS, and all three fractions were brought up to 5 ml with PBS. Samples were subjected to a second ultracentrifugation at 110,000 ϫ g for 70 min. The three resulting pellets were collected and stored at Ϫ80°C. All centrifugations were performed at 4°C in an SW55 Ti rotor.

saPMCA Amplification
To determine the presence of PrP TSE , EV preparations from different sources (source of PrP TSE ) were resuspended into 10% WT-MoBHs (source of PrP C ) that were prepared as indicated above. The volume of 10% WT-MoBH used to resuspend the EV pellets is specified for each individual experiment under "Results." Furthermore, samples were aliquoted into 0.2-ml PCR tubes containing zirconia/silica beads of 1 mm in diameter (Biospec Products Inc., Bartlesville, OK). Samples were amplified by 48 cycles (one round) of incubation at 37°C, followed by a 20-s pulse of sonication at power 4 in a Q700MPX microplate horn sonicator (QSonica, Newtown, CT) (69,70). After each round, sample aliquots were mixed 1:1 or 1:3, as indicated below for each experiment, with 10% WT-MoBH to perform the next round of saPMCA. The number of rounds varied for each experiment and is indicated in the appropriate sections.

Proteinase K (PK) Digestion
Aliquots of 9 l of samples subjected to saPMCA were treated with 20 g/ml PK (Novagen, Darmstadt, Germany) for 1 h at 37°C with agitation at 450 rpm. PK treatment was stopped by sample denaturation in NuPAGE LDS sample buffer (Invitrogen) at 100°C for 10 min.

Methanol Precipitation
Where applicable, samples were mixed with 5 volumes of prechilled 100% methanol and incubated overnight at Ϫ20°C. Samples were centrifuged at 25,000 ϫ g for 30 min in a FA45-24-11 rotor and resuspended in 1% Sarkosyl.

Nanoparticle Tracking Analysis (NTA)
The EV pellet obtained from the plasma of a healthy FVB mouse after purification with ExoQuick TM was resuspended in 200 l of PBS, and the suspension was diluted 1,000-fold prior to being analyzed. Alternatively, the EV pellet obtained as above, was further purified through a 30% sucrose cushion as described under "Exosome Purification Using a 30% Sucrose Cushion." The three fractions collected (top, middle, and bottom) were resuspended in 700 l of PBS prior to being analyzed. The particle hydrodynamic diameter of the EVs was measured for 90 s using a NanoSight LM10 system (Malvern, Worcestershire, UK). The Nanoparticle Tracking Analysis 2.3 analytical software version was used for capturing and analyzing the data. Particle number per ml and standard deviation were calculated from three measurements from the same sample.

saPMCA Detection of PrP TSE in EVs Isolated from Conditioned Media Collected from TSE-infected Cells-We first
investigated the presence of PrP TSE in EVs isolated from conditioned media collected from Mo-vCJD/SP-SC and OF1-BMS and corresponding uninfected cells (NB/SP-SC and BMS, respectively) used as control. EVs were isolated from 5 ml of conditioned media with ExoQuick TM -TC as described under "Experimental Procedures" (EV Isolation from Conditioned Media by ExoQuick TM -TC, Optimized for Culture Media and Urine Samples (System Biosciences, Mountain View, CA)). EV pellets from spleen-derived and bone marrow-derived cells were resuspended in 250 and 200 l, respectively, of 10% WT-MoBH. Samples were amplified by saPMCA through 48 cycles (one round). After each round, aliquots of 30 l were mixed 1:1 with 10% WT-MoBH to perform the next round of saPMCA. Additionally, aliquots of 9 l of amplified material were taken from each sample and processed for the detection of PrP TSE by PK digestion followed by Western blotting as under "Experimental Procedures." EVs from 5 ml of conditioned media contained sufficient amounts of PrP TSE to trigger PrP C conversion in saPMCA reactions, validating the applicability of this approach to detect PrP TSE in these preparations (Fig. 1). One round of saPMCA led to PrP TSE detection in samples generated from Mo-vCJD/ SP-SC cells (Fig. 1A), whereas two rounds were required to detect this protein in samples purified from OF1-BMS cells (Fig. 1C). The difference in number of rounds required for each cell model, which differ in their tissue of origin, might be due to variations in PrP TSE levels in the EV preparations and/or in the number of EVs secreted by the cells. Alternatively, it may reflect differences in the conversion efficiency of the two strains, Mo-vCJD and Fukuoka-1.
Detection of PrP TSE in Uninfected Human and Mouse Plasma Samples Supplemented with EVs from Conditioned Media of TSE-infected Cells-Earlier studies have shown that some blood components impair PMCA amplification (24,25). This inhibition can be overcome by performing several rounds of saP-MCA. Under these conditions, inhibitors are diluted after each round, at the same time that new PrP TSE is generated. This approach enabled PrP TSE detection as follows: in buffy coat samples from 263K-infected hamsters during the preclinical and clinical phases of prion disease (24,25); in plasma preparations from preclinical and clinically sick scrapie-affected sheep, CWD-infected white-tailed deer (28), and from clinically ill mice infected with mouse-adapted BSE (71); in blood leukocytes from clinically sick VRQ/VRQ scrapie-affected sheep (26); and in whole blood from clinical mice infected with scrapie (21). To evaluate the effect of plasma on the detection of EV-associated PrP TSE by saPMCA, EVs isolated from conditioned media of infected cells as described above were added to 250 l of human plasma obtained from a healthy donor. Human plasma was used because of large volumes available to ensure experimental consistency during the study. EVs were re-isolated from EV-supplemented plasma samples with Exo-Quick TM as described under "Experimental Procedures" ("EV Isolation from Plasma by ExoQuick TM , Optimized for Serum or Ascites (System Biosciences)"). Re-isolated EVs were thawed and mixed with 250 l of 10% WT-MoBH. In parallel, EV samples that were not added to plasma were mixed with 250 l of 10% WT-MoBH and used as positive controls for saPMCA. Four aliquots of 58 l were prepared from control and experimental samples and amplified by saPMCA as described under "Experimental Procedures." Samples were diluted 1:1 between saPMCA rounds.
Although PrP TSE was readily demonstrated in EVs from the conditioned media of Mo-vCJD/SP-SC cells after one round of saPMCA ( Fig. 2A), a high unspecific signal background prevented detection of PrP TSE in plasma samples supplemented . EV pellets were mixed with 250 and 200 l, respectively, of 10% wild-type mouse brain homogenate (used as source of PrP C ). Aliquots of 9 l were taken from each sample and used as controls not subjected to saPMCA (Ϫ). The remaining BMS and SP-SC samples were split into 3 and 4 aliquots, respectively, and amplified by saPMCA (ϩ). PrP TSE was specifically detected in EV samples from Mo-vCJD/SP-SC cells (A) and from OF1-BMS cells (C) after one and two rounds of saPMCA, respectively, but not in samples from uninfected NB/SP-SC (B) and BMS (D) cells. All samples were treated with 20 g/ml of proteinase K, resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specific antibody 6D11 (Covance). Molecular mass is shown on the right.
with EVs isolated from the conditioned media of infected cells (Fig. 2B). Nevertheless, dilution and amplification of these samples for three additional rounds of saPMCA resulted in the reliable detection of PrP TSE (Fig. 2A). Similar findings were obtained when EVs, prepared from the conditioned media of Fukuoka-1-infected OF1-BMS cells, were added to plasma collected from uninfected wild-type FVB mice, re-isolated, and used in saPMCA (Fig. 3).
Presence of IgG in Plasma EV Preparations Disables Detection of PrP TSE after the Initial Rounds of saPMCA-IgG is one of the most abundant proteins in plasma, and its presence in samples is a recognized problem in Western blotting applications due to reaction with secondary antibodies. We observed high background signal when performing Western blotting analysis of samples after initial rounds of saPMCA that were seeded with plasma EV preparations. Therefore, we attempted to overcome this problem by using the mouse Trueblot HRP-conjugated anti-mouse IgG (Rockland) secondary antibody, which does not detect the reduced and SDS-denatured forms of IgG, instead of the commonly used secondary HRP-conjugated antimouse IgG (Kirkegaard & Perry) antibody. EVs were isolated with ExoQuick TM from 250 l of plasma samples from two terminally ill FVB mice infected with Mo-vCJD. EV pellets were resuspended in 250 l of 10% WT-MoBH and amplified by saPMCA. Selected saPMCA-amplified plasma EV samples corresponding to rounds one and two were treated with PK, subjected to electrophoresis, and immunoblotted with either antibody (Fig. 4A). The antibody from Kirkegaard & Perry revealed the presence of IgG and other reactive bands, in all samples from the two mice after one round of saPMCA. The intensity of the signal significantly decreased after two rounds demonstrating that cycles of dilution/conversion efficiently eliminated unspecific proteins, including IgG, from the samples. Bands corresponding to IgG were not detected with the antibody from Rockland after rounds one and two. To demonstrate the appropriate binding to the primary antibody 6D11, which is specific to PrP, antibodies from both Kirkegaard & Perry and Rockland were used to reveal PrP TSE in Western blotting of samples subjected to five rounds of saPMCA. Three bands corresponding to di-, mono-, and unglycosylated PrP TSE were observed with either antibody in samples treated with PK; however, the intensity of the signal obtained with the antibody from Rockland was significantly lower, and longer exposure times were necessary to obtain results comparable with those generated with the antibody from Kirkegaard & Perry (Fig. 4B).
Isolation and Characterization of Plasma EVs, PrP TSE Associates with Plasma EVs-To investigate association of PrP TSE with plasma EVs, we took advantage of the flotation properties of nanovesicles on a 30% sucrose cushion. Ultracentrifugation of EVs in the presence of 30% sucrose eliminates nonspecifically associated proteins, or protein aggregates, which are sedimented by centrifugation but do not float on a sucrose gradient and allow isolation of highly purified exosomes (68). Two 500-l aliquots of pooled plasma from clinically sick FVB mice infected with 10 Ϫ1 and 10 Ϫ2 dilutions of Mo-vCJD were processed with ExoQuick TM to isolate EVs. The pellet from 1 aliquot was stored at Ϫ80°C until analysis and was labeled as "infected non-sucrose." The pellet from the second aliquot was processed through a 30% sucrose cushion as described under "Experimental Procedures." Three fractions were collected after ultracentrifugation as follows: a thin infected top layer of ϳ0.2 ml of white material of lipidic appearance; the infected middle layer containing the PBS/sucrose interface and the sucrose layer; and the infected bottom pellet, which was not visible. All three fractions were brought up to 5 ml of PBS and subjected to a second ultracentrifugation. Plasma samples from uninfected FVB mice were processed similarly and used as negative controls ("uninfected non-sucrose" and "uninfected top, middle, and bottom"). Infected and uninfected "non-sucrose" fractions were resuspended in 250 l of 10% WT-MoBH; infected and uninfected "sucrose" fractions were resuspended in 125 l of 10% WT-MoBH. All samples were subjected to various rounds of saPMCA.
Samples were evaluated for the presence of PrP TSE by Western blotting with the anti-PrP primary antibody 6D11 and mouse Trueblot secondary antibody (Fig. 5). This allowed PrP TSE detection in plasma EVs at early rounds of saPMCA without IgG interference. Under these conditions, PrP TSE was specifically detected in "non-sucrose" plasma exosome frac-   2 and 3,  and B, lanes 3-6) or added to 250 l of FVB mouse plasma. Samples added to plasma were subjected to repeated EV extraction with ExoQuick TM , and the pellets were mixed with 150 l (A, lanes 4 and 5, and B, lanes 7 and 8), 250 l (A, lanes 6 -9, and B, lanes 9 -12), and 1,250 l of WT-MoBH (A, lanes 10 and 11, and B, lanes  13 and 14). Each sample was split into aliquots prior to saPMCA (ϩ). The presence of heavy background signal in EVs re-isolated from plasma prevented PrP TSE by Western blotting detection after one round of saPMCA. Dilution of samples in three and four additional rounds allowed reliable detection of PrP TSE (A and B, respectively). Samples were treated with 20 g/ml of proteinase K (PK), resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specific antibody 6D11 (Covance). ϪPK, WT-MoBH not treated with PK. Molecular mass (M) is shown on the left. Samples were diluted 1:5 between saPMCA rounds.

FIGURE 4. Presence of IgG in plasma-derived EV preparations.
Western blotting analysis of the initial rounds of saPMCA showed high background that prevented the detection of PrP TSE . A, presence of IgG was evaluated by assessing sample reactivity with two secondary antibodies: HRP-conjugated goat anti-mouse IgG (Kirkegaard & Perry antibody (Ab)) and mouse Trueblotா HRP-conjugated rat anti-mouse IgG (Rockland antibody), which does not detect the reduced SDS-denatured forms of IgG. saPMCA-amplified plasma EVs from terminally ill FVB/NCr mice infected with Mo-vCJD after rounds one and two were digested with proteinase K and separated on SDS-PAGE prior to immunoblotting with either antibody (A, left and right panels, respectively). Immunoblotting with the antibody from Kirkegaard & Perry revealed the presence of IgG in all aliquots from both mice after one round of saPMCA. The intensity of the signal significantly decreased after two rounds of saPMCA, demonstrating that cycles of dilution/conversion effectively eliminate IgG in plasma EV-seeded samples. No signal was detected with mouse Trueblotா antibody. B, same plasma EV preparations were probed with the PrP-specific antibody 6D11 (Covance) as the primary antibody was followed by secondary antibodies from either Kirkegaard & Perry or Rockland. Molecular mass is shown on the right. Note the difference in the intensity signals between the antibodies used from Kirkegaard & Perry and Rockland, with the antibody from Kirkegaard & Perry giving significantly better signal readout. Lanes 1-3 represent sample triplicates.
tions from infected mice after just one round of saPMCA (Fig.  5A, infected non-sucrose). A second round of saPMCA showed the presence of PrP TSE in the pellet fraction obtained after centrifugation through a sucrose cushion (Fig. 5B, lanes 3 and 4). In addition, PrP TSE was detected in the "top" layer ( Fig. 5B, lanes 1  and 2), which represents a fraction of co-purified PrP TSE that is likely to be associated with lipids, very low density and low density lipoproteins (72), and/or because the ExoQuick TM protocol does not include a filtration step, probably with membrane fragments. Most importantly, PrP TSE was detected in the "middle" fraction ( Fig. 5B, lanes 5 and 6), containing plasma exosomes. No PrP TSE was detected in corresponding fractions from plasma EV preparations from uninfected mice (Fig. 5, A,  lanes 5-8, and B, lanes 7-12). Samples were diluted 1:1 between rounds.
Exosomal Marker Hsp70 Associates with Plasma EVs-To characterize EV preparations, we analyzed the presence of the exosomal marker Hsp70 and the absence of the Golgi marker GM130. EVs were isolated from 500 l of plasma from a healthy FVB mouse using ExoQuick TM . The EV pellet was subjected to ultracentrifugation through a 30% sucrose cushion and repeated ultracentrifugation as described above. The three pellets, representing sucrose top, middle, and bottom fractions, were resuspended in EVP lysis buffer to solubilize membrane proteins. Proteins were concentrated by methanol precipitation and resuspended in 1% Sarkosyl in PBS. For comparison, EVs were also isolated from the conditioned media of NB/SP-SC cells with ExoQuick TM -TC, resuspended in EVP lysis buffer, methanol-precipitated, and resuspended in 1% Sarkosyl in PBS ("cell EVs"). This preparation was used as positive control for Hsp70. Additionally, NB/SP-SC cells were lysed in EVP lysis buffer ("NB/SP-SC cell lysate") and used as control for GM130. The same amount of total protein (12 g) was loaded per sample. Western blotting analysis of samples obtained from plasma, conditioned media, and cell lysates revealed the presence of Hsp70 in the conditioned media sample (cell EVs) and sucrose middle plasma fraction (Fig. 6A, lanes 1 and 4, respectively) but not in sucrose top and bottom plasma fractions obtained after ultracentrifugation (Fig. 6A, lanes 3 and 5), thus confirming that samples prepared from plasma using ExoQuick TM contained exosomes. As expected, the Golgi marker GM130 was exclusively detected in cell lysates (Fig. 6B, lane 1) but was absent in samples prepared from conditioned media and from sucrose top, middle, and bottom fractions (Fig. 6B, lanes 2-5). The specificity of the immunoreactive bands was confirmed by the absence of signal while probing the samples with the secondary rabbit Trueblot antibody only (data not shown). EV samples were prepared using Exo-Quick TM (System Biosciences) from 500 l of plasma from clinically sick or uninfected control mice as described in the text. A, EV pellets were mixed into 250 l of 10% wild-type mouse brain homogenate (WT-MoBH), split into 4 aliquots, and subjected to one round of saPMCA (non-sucrose). B, EV pellets were used to purify exosomes by ultracentrifugation on a 30% sucrose cushion. Three fractions were collected (top, middle (containing exosomes), and bottom) and subjected to a second round of ultracentrifugation. Pellets were mixed into 125 l of 10% WT-MoBH, split into 2 aliquots, and subjected to two rounds of saPMCA. Aliquots of 9 l were collected from each sample, treated with 20 g/ml proteinase K, resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specific antibody 6D11 (Covance) and mouse Trueblotா (Rockland) secondary antibody.
ϪPK, WT-MoBH not treated with PK. Molecular mass is shown on the right. Samples were diluted 1:1 between rounds.

Detection of Particle Hydrodynamic Diameter in EV Preparations
Purified by ExoQuick TM -To identify the particle hydrodynamic diameter of EVs, samples prepared from the plasma of healthy FVB mice were subjected to NTA. The vesicle hydrodynamic diameter ranged from 50 to 300 nm, with an average diameter of 114 nm, in a sample with a vesicle count of 5.46 ϫ 10 8 particles/ml (Fig. 7A). Established EV hydrodynamic diameter was within the range reported for human plasma EVs (73). Next, we evaluated the presence of EVs in the three fractions collected following centrifugation through a 30% sucrose cushion. To this end, EVs were isolated from the plasma of healthy FVB mice with ExoQuick TM and further purified through a 30% sucrose cushion as described above. The top, middle, and bottom fractions were resuspended in PBS and subjected to NTA. We identified the presence of EVs in all fractions analyzed, albeit with differences in particle concentration and hydrodynamic diameter. The top fraction was characterized by the presence of a single population of EVs ranging from 38 to 350 nm and an average hydrodynamic diameter of 103 nm (Fig. 7B). The middle fraction contained EVs with a hydrodynamic diameter range of 25 to 450 nm and a major particle population of 102 nm. Two other minor peaks were detected in this preparation showing average hydrodynamic diameters of 124 and 145 nm (Fig. 7C). The bottom fraction contained EVs of 40 to 500 nm in diameter. Various peaks could be identified in this fraction with average hydrodynamic diameters of 87, 114, 139, and 170 nm (Fig. 7D). The particle concentration also varied between fractions, with the bottom fraction containing the lowest number of particles, 7.2 Ϯ 0.21 ϫ 10 8 particles/ml, followed by the top fraction with 11.6 Ϯ 0.21 ϫ 10 8 particles/ml, and the middle fraction with 17.3 Ϯ 1.33 ϫ 10 8 particles/ml.
Detection of PrP TSE in Plasma EVs Isolated from Symptomatic Mo-vCJD-infected FVB Mice-After demonstrating the efficiency of ExoQuick TM to isolate PrP TSE -containing EVs and the potential of saPMCA to detect PrP TSE in plasma EV preparations (Fig. 5), we aimed to confirm the presence of PrP TSE in plasma preparations from a larger number of samples collected from terminally sick mice infected with Mo-vCJD.
EVs were isolated from 200 and 250 l of plasma from Mo-vCJD-infected FVB mice and appropriate uninfected controls, using ExoQuick TM as described under "Experimental Procedures." Samples were resuspended in an equal volume of 10% WT-MoBH, split into 4 aliquots, and subjected to multiple rounds of saPMCA. Samples were diluted 3-fold between saPMCA rounds. Results of PrP TSE detection, starting from round five, in which the first positive samples were clearly identified, are summarized in Table 1 and Fig. 8. PrP TSE was detected in all plasma EV pellets from infected mice in which TSE had been biochemically confirmed. Samples from mouse 4, which received a 10 Ϫ7 dilution of Mo-vCJD, were negative through all seven rounds. In agreement with this result, mouse 4 showed no clinical signs of TSE and tested negative for PrP TSE in the brain by Western blotting. The calculated LD 50 value for the Mo-vCJD brain homogenate, used for inoculation, was estimated to be 10 Ϫ6. 3 . The other three mice in the group infected with a 10 Ϫ7 dilution exhibited clinical signs and tested positive for PrP TSE in the brain by Western blotting. Samples from uninfected control mice remained negative for all rounds of saPMCA (Fig. 8,  FVB-I and FVB-II). Likewise, PrP TSE was specifically detected in plasma EVs isolated from FVB mice inoculated with Fukuoka-1; samples collected from noninfected mice remained negative for five rounds of saPMCA (data not shown).
Detection of PrP TSE in EVs Isolated from Presymptomatic and Symptomatic Mo-vCJD-infected C57BL Mice-Plasma EV pellets were obtained from groups of Mo-vCJD-infected C57BL mice that were euthanized at 6,9,12,15, and 20 wpi and at clinical stage of TSE (ϳ23 wpi) and from uninfected control mice. Samples were evaluated for the presence of PrP TSE after several rounds of saP-MCA as described above. The results of this experiment are summarized in Table 2 and Fig. 9. All mice had tested positive for PrP TSE in the brain by Western blotting (Fig. 10). Four rounds of saPMCA reliably revealed the presence of PrP TSE in all aliquots of plasma EVs obtained from mice euthanized at 20 wpi and during

in plasma-derived EV preparations isolated from clinically sick Mo-vCJD-infected FVB mice
The presence of PrP TSE was tested in 4 aliquots of EV preparations from individual or pooled plasma samples obtained from terminally ill Mo-vCJD-infected mice. All infected mice had tested positive (ϩ) for PrP TSE in the brain by Western blotting, except mouse 4 (Ϫ). EVs were isolated from 200 l of plasma by ExoQuick TM (System Biosciences). EV pellets were mixed with 200 l of 10% wild-type mouse brain homogenate and split into 4 aliquots that were subjected to up to seven rounds of saPMCA. A summary of the data from rounds 5 to 7 is presented. Background interference prevented identification of PrP TSE -positive samples prior to round five. Presence of PrP TSE was evaluated by Western blotting using the PrP-specific antibody 6D11 (Covance). FVB-I and FVB-II indicate plasma samples from FVB control mice.   OCTOBER 17, 2014 • VOLUME 289 • NUMBER 42 the clinical phase. Moreover, PrP TSE was demonstrated in at least 1 of 4 aliquots from mice from earlier disease stages starting from 6 wpi, except for mouse 1. One additional round of conversion increased the number of positive aliquots from animals 3, 5, and 12, euthanized at 6, 9, and 15 wpi. Mouse 1 again tested negative for PrP TSE . Aliquots from samples generated from uninfected mice remained negative for all five rounds of saPMCA. Conclusively, the presence of PrP TSE was demonstrated in plasma EVs from Mo-vCJD-infected mice during the preclinical phase, as early as 6 wpi. This finding is in agreement with the work of Tattum et al. (21) showing detection of PrP TSE , after four rounds of saPMCA, in whole blood collected at 60 days post-inoculation from scrapieinfected CD1 mice.

DISCUSSION
The finding of four human vCJD infections related to blood transfusion (4) highlights the need to develop a method that FIGURE 9. Detection by saPMCA of PrP TSE in plasma EV preparations isolated from presymptomatic and symptomatic Mo-vCJD-infected C57BL mice. EVs were isolated with ExoQuick TM (System Biosciences) from 200 and 250 l of plasma collected from presymptomatic and symptomatic C57BL mice intracerebrally injected with a 1% Mo-vCJD-infected brain homogenate. EV pellets were mixed with 200 and 250 l, respectively, of 10% wild-type mouse brain homogenate (WT-MoBH), split into 4 aliquots, and amplified by saPMCA. After each round, aliquots of 20 l were taken from each sample, mixed with 40 l of 10% WT-MoBH, and amplified in a new round of saPMCA; additionally, aliquots of 9 l were collected from each sample, treated with 20 g/ml of proteinase K (PK), resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specific antibody 6D11 (Covance). The figure illustrates the presence/absence of PrP TSE signal in each of 4 sample aliquots from each mouse after five rounds of saPMCA. C57BL-I and C57BL-II, samples from uninfected wild-type mice used as negative control; ϪPK, WT-MoBH not treated with PK. Molecular mass is shown on the right. . Detection of PrP TSE in brain tissue from presymptomatic and symptomatic Mo-vCJD-infected C57BL mice. 10% mouse brain homogenates were treated with 20 g/ml of proteinase K, resolved by SDS-PAGE, and subjected to Western blotting using the PrP-specific antibody 6D11 (Covance). Molecular mass is shown on the left.

TABLE 2 Detection by saPMCA of PrP TSE in plasma EV preparations isolated from Mo-vCJD-infected C57BL/6 (C57CL) mice, pre-symptomatic and clinical phase
The presence of PrP TSE was tested after four to five saPMCA rounds in 4 aliquots of EV preparations from individual plasma samples obtained at various post-inoculation times from Mo-vCJD-infected C57BL mice. All infected mice had tested positive for PrP TSE in the brain. Plasma samples from uninfected mice were used as negative controls (C57BL-I and C57BL-II). EVs were isolated from 200 or 250 l of plasma by Exo-Quick TM (System Biosciences). EV pellets were mixed with 200 or 250 l of 10% wildtype mouse brain homogenate, respectively, and split into 4 aliquots that were subjected to saPMCA. Repeated rounds of saPMCA were performed. The presence of PrP TSE was evaluated by Western blotting using the PrP-specific antibody 6D11 (Covance). Boldface indicates plasma extracellular vesicle samples that tested positive for PrP TSE in all 4 aliquots. can reliably identify preclinical cases to minimize the risk of iatrogenic disease transmission via blood-derived products. Experimental detection of PrP TSE in animal cerebrospinal fluid (CSF) (29), whole blood (21,22), plasma (27)(28)(29)(30)71), buffy coat (24 -26), and urine (74) and in human CSF (12,75), whole blood (6 -8), and urine (76,77) has been achieved by different methods that rely on the concentration or amplification techniques to bring PrP TSE levels to the detection threshold of biochemical assays. Although the presence of PrP TSE in blood exosomes has been suggested previously (48), biochemical detection has been complicated by the low levels of PrP TSE in blood and the concomitantly large volumes required for exosome isolation by standard methods. Traditionally, exosome isolation has been achieved by a series of differential centrifugation and filtration steps from large sample volumes (68). This approach has been combined with saPMCA to demonstrate the presence of PrP TSE in exosomes isolated from the conditioned media of cell cultures infected with M1000, a mouse-adapted sCJD strain (52). In the context of disease diagnosis using body fluids, limitations exist as to the sample volume available for evaluation. In recent years, new reagents have become available for isolating exosomes from small volumes expected in such clinical applications. Two of these reagents, Exo-Quick TM -TC and ExoQuick TM , have been repeatedly shown to successfully isolate exosomes from conditioned media and biological fluids for various downstream applications (78 -82). By combining extracellular vesicle isolation using ExoQuick TM with PrP TSE amplification by saPMCA, we were able to demonstrate the presence of PrP TSE in plasma samples collected from preclinical and sick mice infected with Mo-vCJD, and we showed for the first time its association with blood-circulating EVs. Biochemical characterization of EV preparations obtained from plasma and conditioned media showed the presence of the exosomal marker Hsp70 and the absence of the Golgi marker GM130, thus demonstrating the efficiency of ExoQuick TM reagents to purify EVcontaining exosomes. Moreover, processing of EVs by ultracentrifugation through a 30% sucrose cushion, coupled with saPMCA amplification, allowed the detection of PrP TSE in EVs that were confirmed to carry the exosomal marker Hsp70, further indicating the possible association of PrP TSE with these vesicles. There are three types of EVs released by the cell that differ in their biogenesis and that can be classified as follows: (i) apoptotic vesicles (50 -5000 nm in diameter) released by apoptotic cells; (ii) exosomes (40 -200 nm) released upon fusion of multivesicular bodies with the plasma membrane, and (iii) microvesicles (50 -1000 nm) that directly bud from the plasma membrane (37,48,83). Because some of these vesicles overlap in size, it is difficult to separate them by the current isolation methods. In particular ExoQuick isolates EVs ranging in size from 17 to 200 nm. Nevertheless, the demonstration of the exosomal marker Hsp70 exclusively in the middle fraction, where exosomes fractionate following centrifugation in a 30% sucrose cushion, confirms the presence of these vesicles in our preparations. The fact that PrP TSE was also identified after saPMCA in the top and bottom fraction where other EVs have been detected by NTA suggests that it can associate with other classes of EVs, although this conclusion requires further investigation because, as mentioned above, the top and bottom fraction may contain lipoprotein-associated PrP TSE and PrP TSE aggregates, respectively, not associated with EVs. Interestingly, on the bases of the accumulated evidence that PrP C is present at the exosomal membrane (46 -50, 53, 58, 84 -87), this protein is being used to characterize exosomal preparations (88). Additionally, we demonstrated that EV isolation can be scaled down to volumes compatible with clinical applications. Two hundred microliters of plasma provided enough material to allow PrP TSE detection by saP-MCA in murine samples. The challenge will be to detect PrP TSE in similar volumes of human plasma. Western blotting analysis of plasma EVs, isolated with ExoQuick TM , revealed the presence of heavy and light chains of IgG in our samples. These findings are in line with various studies that showed association of immunoglobulin light chains (89) and various types and subtypes of heavy chains with exosomes isolated from tumors and body fluids (89 -92). The presence of IgG in EVs isolated using ExoQuick TM hindered the detection of PrP TSE in the initial rounds of saP-MCA by Western blotting due to a high nonspecific background signal. Serial dilution/amplification significantly reduced IgG, effectively eliminating the signal interference and revealing the presence of misfolded PrP TSE . Detection of PrP TSE in plasma EVs after just one round of saPMCA was achieved with a secondary antibody that does not recognize heavy and light chains of IgG under reduced conditions. However, this antibody was only used in our confirmatory assays due to practical reasons, i.e. high cost and low reading signal in Western blotting. Additional methods to remove IgG from plasma sample preparations are currently under evaluation; we expect that these approaches will reduce the number of rounds required to detect PrP TSE . The number of amplification rounds needed to identify PrP TSE varied between animals infected with the same TSE agent and between cell cultures infected with Mo-vCJD or Fukuoka-1. These findings suggest that variability exists between individual mice as far as the quantities of PrP TSE present in plasma at the terminal stages of disease as well as differences in conversion efficiencies between TSE strains. However, we cannot exclude differences in the PrP TSE replication capacity of our cell models.

Blood collection time post-inoculation
In summary, our experiments provide a valuable foundation for the development of new diagnostics and potential targets for TSE treatment. Moreover, our data provide evidence that a fraction of blood-circulating PrP TSE co-localizes with exosomes. This finding opens new avenues for further TSE research. Because exosomes are known to not only participate in cell to cell communication processes (34,58,59,93), but to also cross the blood-brain barrier (94,95), association of PrP TSE with exosomes may serve as a platform for TSE spread from the periphery to the CNS (47,56,58). Additionally, we are further characterizing PrP TSE -bearing microvesicles with the goal of identifying the cellular origin of this protein in blood. This information may advance the development of new strategies to ensure the safety of blood-and plasma-derived products.