JGV Papers in Press. Published March 24, 2015 as doi:10.1099/vir.0.000117
Journal of General Virology Detection of exosomal prions in blood by immunochemistry techniques --Manuscript Draft-Manuscript Number:
JGV-D-14-00185R2
Full Title:
Detection of exosomal prions in blood by immunochemistry techniques
Short Title:
Exosomal prions in blood
Article Type:
Short Communication
Section/Category:
TSE Agents
Corresponding Author:
Maurizio Pocchiari Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità Roma, ITALY
First Author:
Francesca Properzi
Order of Authors:
Francesca Properzi Mariantonia Logozzi Hanin Abdel-Haq Cristina Federici Luana Lugini Tommaso Azzarito Ilaria Cristofaro Daniela di Sevo Elena Ferroni Franco Cardone Massimo Venditti Marisa Colone Emmanuel Comoy Valérie Durand Stefano Fais Maurizio Pocchiari
Abstract:
In most forms of prion diseases blood is infectious, but the detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of exosome on sucrose gradient was necessary for removing plasma immunoglobulins, which interfere with PrPTSE masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.
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Detection of exosomal prions in blood by immunochemistry techniques
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Running title: Exosomal prions in blood
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Francesca Properzi, Mariantonia Logozzi, Hanin Abdel-Haq, Cristina Federici, Luana Lugini, Tommaso
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Azzarito, Ilaria Cristofaro, Daniela di Sevo, Elena Ferroni, Franco Cardone, Massimo Venditti, Marisa Colone,
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Emmanuel Comoy, Valérie Durand, Stefano Fais and Maurizio Pocchiari
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Affiliations
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Francesca Properzi, Hanin Abdel-Haq, Ilaria Cristofaro, Daniela di Sevo, Elena Ferroni, Franco Cardone,
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Massimo Venditti, and Maurizio Pocchiari, Department of Cell Biology and Neurosciences, Istituto
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Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.
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Mariantonia Logozzi, Cristina Federici, Luana Lugini, Tommaso Azzarito, and Stefano Fais, Department of
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Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, 00161 Rome, Italy;
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Marisa Colone, Department of Technologies and Health, Istituto Superiore di Sanità, 00161 Rome, Italy
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Emmanuel Comoy and Valérie Durand, Institute of Emerging Diseases and Innovative Therapies, CEA,
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Fontenay-aux-Roses, France
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Corresponding author
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Maurizio Pocchiari
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Email: [email protected]
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Telephone: +39 06 49903203
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Fax: +39 06 49903805
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Word Count Summary: 93
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Tables:
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Suppl. Figures:
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Summary paragraph
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In most forms of prion diseases blood is infectious, but the detection by immunochemistry techniques of
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the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We
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developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the
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finding that PrPTSE is associated with plasma exosomes. However, further purification of exosome on
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sucrose gradient was necessary for removing plasma immunoglobulins, which interfere with PrPTSE masking
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its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with
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exosomes.
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Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal neurodegenerative disorders
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affecting humans and animals caused by unique infectious particles (prions), which are mainly composed
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by misfolded forms of the cellular prion protein (PrPC). PrPC is constitutively expressed, but converts to the
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misfolded noxious structure (PrPTSE) either spontaneously or upon exposure to prions (Weissmann, 2004).
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Prions are efficiently transmitted via blood in most animal models of TSE diseases (Brown et al., 2001)
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(Houston et al., 2008) and in humans with variant and possibly sporadic CJD (Llewelyn et al., 2004) (Wroe
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et al., 2006) (Puopolo et al., 2011) (Douet et al., 2014). Prion infectivity in blood is associated with both
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cellular and non cellular components (Gregori et al., 2006) with estimated titres in plasma ranging from 1
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to 10 infectious unit/ml at disease onset (Brown et al., 2001) (Douet et al., 2014). Although plasma has
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been shown to carry infectivity, the identification of PrPTSE in infected animals and humans has been elusive
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and limited to the use of highly sensitive (Castilla et al., 2005) (Saá et al., 2006) (Pan et al., 2007) (Orrú et
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al., 2011) or complex (Edgeworth et al., 2011) methodologies. Because blood components likely interfere
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with PrPTSE detection (Hartwell et al., 2005) (Abdel-Haq, 2014), we thought to bypass this difficulty by
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looking at plasma exosomes. Exosomes are nanovesicles that carry RNAs, proteins, lipids, other metabolites
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(Théry et al., 2009) (Fais et al., 2013), PrPC and, in prion-infected hosts, PrPTSE and infectivity (Fevrier et al.,
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2004) (Alais et al., 2008) (Coleman et al., 2012) (Saá et al., 2014).
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We first collected blood from intracerebrally 263K scrapie-infected hamsters (n=125) (Kimberlin and
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Walker, 1977) or from uninfected and healthy animals as controls. Plasma was separated from blood cells
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by centrifugation at 5,000 g for 30 minutes at room temperature. Single plasma fractions from infected
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hamsters were pooled to a final volume of 250 ml and used for the measurement of infectivity and
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purification of exosomes for the detection of PrPTSE by immunochemistry assays (Fig. 1). Bioassay was
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performed by intracerebral inoculation of 124 six weeks-old female Syrian hamsters with 50 µl of thawed
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plasma, under anesthesia with ketamine/xylazine (75 and 10 mg/kg respectively) mixture. Animals were
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housed in accordance to the national guidelines in level-3 animal care facilities (agreement numbers A 92-
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032-02 for animal care facilities, 92-189 for animal experimentation, CEA, France) and regularly monitored.
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Animals with clinical signs or with no disease up to 600-700 days were euthanized, the brain removed and
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tested for the presence of PrPTSE by the ELISA test as previously described (Barret et al., 2003). Briefly, PrPTSE
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was extracted from brains by the Bio-Rad purification kit (USA), digested with proteinase K, and added into
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anti-PrP SAF53 (Bertin Pharma, France) monoclonal antibody (mAb)-coated ELISA wells. Bound PrPTSE was
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detected by incubation with the 11C6 (Bertin Pharma, France) anti-PrP mAb coupled with
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acetylcholinesterase, and revealed using the Ellman substrate at 414 nm optical wavelength.
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Eighteen animals resulted positive for scrapie (time to death, mean, 265 days; s.e.m., 23 days) with a
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corresponding infectivity titre of 3.1±0.7 infectious dose (ID) per ml (Supplementary Fig. 1), in line with
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previously published results on plasma samples (Gregori et al., 2006) (Brown et al., 2001). Infectivity titre
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(ID) was calculated by the limiting dilution method according to the Poisson distribution (Gregori et al.,
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2006).
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From the same pool of plasma, exosomes were isolated from 5 ml aliquots of control and infectious plasma
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pool by a previously published protocol (Caby et al., 2005). Briefly, exosome plasma pellets were prepared
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by serial centrifugations; the resulting pellets were washed in PBS, filtered through a 0.22 μm filter
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(Millipore, USA) and ultracentrifuged at 110,000g for 1 h. The exosome pellets were re-suspended in PBS,
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lysis buffer or in 2.5 M sucrose buffer, for further analyses as indicated in Fig. 1. Antibodies used for
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exosomal marker identification were the anti-Flotillin, anti-Tsg101, and anti-mouse CD81 (Santa Cruz,
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Germany). For PrP identification the primary antibodies used were SAF84 and biotinylated-SAF84 (Bertin
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Pharma, France), 3F4 (Millipore, Italy), and D18 (gift from R. A. Williamson, The Scripps, Florida). Western
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blot analyses were performed as described (Lee et al., 2000) and the consistency of these preparations
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confirmed by the detection of exosomal markers (Fig. 2a). Assuming a similar ratio between infectivity and
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PrPTSE molecules in blood and in brain tissue (about 0.1 to 1 pg PrPTSE/infectious unit) (Brown et al., 2001),
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we estimated that 25 µl of exosomal plasma pellets (EPPs), corresponding to 5 ml of the original plasma
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volume and 15.5 IDs, were suitable for detecting PrPTSE by standard western blot analyses (Lee et al., 2000).
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In EPPs from control and prion-infected samples, we found that anti-PrP antibodies recognised the Ig light
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chain (26 kD), and the IgG (53 kD) and IgM (65 kD) heavy chains as previously reported (Hartwell et al.,
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2005). These bands were resistant to an amount (50 µg/ml) of proteinase K (PK) that is largely sufficient to
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remove the bulk of tissue proteins in brain samples (Fig. 2). The atypical bands were seen irrespectively of
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the anti-PrP primary antibody used (Supplementary Fig. 2) and were not detected in the absence of primary
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antibodies (Fig. 2a). They were specific to plasma (Fig. 2a) and observed in EPPs of other species including
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humans (data not shown) and wild type mice (C57BL/10) (Fig. 2a and supplementary Fig. 2). They were also
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reproduced in EPPs of PrP knock-out 129/Ola mice (gift from Dr Di Bari and Dr Agrimi, Istituto Superiore di
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Sanità, Rome, Italy) (Fig. 2a and supplementary Fig. 2) clearly indicating a cross-reaction of the primary
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antibodies with plasmatic PK-resistant material, unrelated to PrP epitopes. Finally, when primary anti-PrP
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antibodies were pre-absorbed with an excess of Syrian hamster 23-231 recombinant PrP (recPrP) (5:1),
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western blot membranes were negative, demonstrating that pre-absorbed primary antibodies are unable
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to bind PK-resistant Igs (IgRES). These data suggest that the levels of endogenous PrPC in plasma (in the
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range of pg/ml) is not sufficient to replace the non-specific binding of largely represented plasma proteins
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(in the range of mg/ml).
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Stringent PK conditions (100 µg/ml), however, digested high molecular weight IgRES bands (Fig. 2a)
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allowing the recognition of three bands of the same molecular weight of full length prion protein with a
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glycoform ratio similar to that observed in hamster brain (Fig. 2a) (Meade-White et al., 2009). We then
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further purified EPPs on a sucrose cushion starting from 15 ml of plasma to verify that these bands
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effectively belong to full-length prion protein. Continuous sucrose gradient for the isolation and purification
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of exosomes was performed as published (Raposo et al., 1996), with some modifications. Briefly, purified
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exosomes were re-suspended in 2 ml of 2.5 M sucrose, 20 mM Hepes/NaOH, pH 7.2. A linear sucrose
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gradient (2.0-0.25 M sucrose) was layered on top of the exosomes suspension in a UltraClear™ tube and
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the sample was centrifuged at 110,000 g for 16 h at 4oC in a Beckman-Coulter (USA) SW41Ti rotor. Gradient
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fractions (12 x 1 ml) were collected from the top of the tube and washed with PBS. Fractions density was
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evaluated using an Abbé Refractometer (Carl Zeiss, Germany).
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Silver
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http://imagej.nih.gov/ij/) showed that after sucrose gradient ultracentrifugation the bulk of the
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immunoglobulins was retained at high-density fractions (1.64 to 1.70 g/ml), while exosomal fractions (1.11
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to 1.13 g/ml) had significantly lower content of IgRES. On sucrose gradient purified EPPs, di-glycosylated
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PrPC bands (37 kD) were detected at densities of 1.13 g/ml (Fig. 3) and disappeared after PK treatment (Fig.
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3). This fraction was also positive on western blot to standard exosomal markers (Tsg101, Flotillin and
staining
after
PAGE
and
quantification
of
bands
using
ImageJ
(NIH,
available
at
7 120
CD81) and negative to ER and nucleus markers (calnexin and nucleoporin) indicating the purity of exosomal
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preparation after sucrose gradient isolation (Fig. 3). As controls, ER and nucleus markers resulted positive in
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the brain homogenate (Fig. 3).
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In infectious EPPs, 37 kD PrP bands were observed in exosomal fractions at densities of 1.13 g/ml (Fig. 3).
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After treatment with PK, PrP-positive bands showed the characteristic shift in molecular weights of PrPTSE
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(27-30 kD) with a glycoform ratio similar to that observed in 263K-infected brain homogenate (Fig. 3). The
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presence of PrPC on plasma exosomes was further ascertained by electron microscopy. Immunoelectron
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microscopy was performed on isolated sucrose gradient fractions of hamster plasma exosomes. A drop of
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exosome suspension was placed onto a formvar-carbon-coated grid. After primary antibody (SAF84)
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incubation exosomes were labelled with anti-rabbit IgG or anti-mouse IgG 5nm diameter-gold particle-
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conjugated antibody (Sigma-Aldrich, USA). Grids were negatively stained with uranyl acetate solution and
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examined with a Philips 208 transmission electron microscope (FEI Company, The Netherlands). In the 1.13
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g/ml fraction there was presence of nanovesicles, which after immunogold staining, were PrP-positive
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particles on the surface of nanovesicles (~50 nm) (Supplementary Fig. 3a and 3b). On these samples,
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nanovesicles of the same size were positive to the standard exosomal marker Rab5b and no PrP staining
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was observed in fractions of lower and higher densities (Supplementary Fig. 3c).
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Finally, to further confirm the presence of prion in plasma exosomes we measured scrapie infectivity.
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However, for this experiment we used the mouse-adapted 139A scrapie strain (Dickinson, 1976), because
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139A, but not 263K infectivity can be quickly and accurately measured by using the End Point Scrapie Cell
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Assay (SCEPA) (Klöhn et al., 2003). Susceptible CAD5 cells (gift from Prof. Charles Weissmann, The Scripps,
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Florida, USA) were seeded in wells of 96-well plate and exposed to 12 or more aliquots of the 139A prion-
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containing samples for 3 days, grown to confluence and split 1:3 three times and 1:10 twice. When the cells
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reached confluence, 25,000 cells were transferred to membranes of ELISpot plates and PrPTSE-containing
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cells were identified as spots using the D18 antibody (Supplementary Fig. 4). A well is considered positive
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when the following two criteria are fulfilled. Firstly, the number of positive spots must be higher then the
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mean plus 5 times standard deviations of unseeded wells (background value). Secondly, prion propagation,
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revealed by an increased number of positive spots after subsequent passages, must be evident. The ID was
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calculated considering the number of negative wells over positive by using a Poisson distribution. A titred
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brain homogenate is used as positive control to monitor variations caused by antibodies. Nanovesicles
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were isolated from 3 ml of plasma of 139A-infected mice at terminal stage. We found 1 of 6 positive wells
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for exosomal samples and 4 of 48 positive wells for the 1:5 dilution of plasma supernatant. Accordingly to
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the Poisson distribution for rare events, the IDs contained in the processed volume (3 ml) were 1.1 for the
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exosomal pellet and 4.3 for plasma supernatant. These findings confirm that in vivo prions are associated to
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nanovesicles similarly to what previously observed in vitro (Fevrier et al., 2004) (Alais et al., 2008) (Coleman
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et al., 2012) (Fauré et al., 2006) (Vella et al., 2007) and that in plasma about 20% of total infectivity is
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associated to exosomes leaving open the issue on how the remaining infectivity is distributed.
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The most important finding of our work is the successful identification of PrP TSE in plasma of scrapie-
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infected hamsters by standard western blot technique performed on exosomal fractions. This result
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enforces previous data showing the presence of PrPTSE in blood exosomes by the protein misfolding cyclic
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amplification (PMCA) method (Saá et al., 2014), in plasma by the PMCA (Castilla et al., 2005) and RT-QuiC
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assays (Orrú et al., 2011) and in human vCJD patients by the solid-state binding matrix assay (Edgeworth et
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al., 2011), but adds the important novel information that PrPTSE, similarly to infectivity (Brown et al., 2001)
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is detectable in blood without using amplification techniques. The detection of PrP TSE by the standard
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western blot technique was only possible after reducing the interference of IgRES by exosomal pellet
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fractionation on a sucrose gradient. IgRES compete for the primary antibody binding with endogenous PrP
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epitopes crucially interfering with PrPTSE detection in blood. This result confirms that blood components
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interfere with prion diagnosis (Hartwell et al., 2005) (Abdel-Haq, 2014) reducing the sensitivity and
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specificity of the various tested assays. Moreover, these findings might explain the discrepancy between
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the excellent performances of tests accomplished on spiked blood and their failure on endogenously
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infected blood that is likely related to different interaction with plasma Igs between exogenous and
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endogenous PrPTSE.
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Acknowledgments
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We thank Dr Anna Ladogana for critical revision of the manuscript, Giuseppe Loreto for the preparations of
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the figures, Dr Agnese Molinari for support with EM, Nick Verity for supplying the SCEPA samples, and Dr
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Alessandra Garozzo for editorial assistance.
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This work was partially supported by the Istituto Superiore di Sanità, Rome, Italy and by the Alliance
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Biosecure Project “Plasmasecure”.
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Figure Legends
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Figure 1
257
Infectivity of plasma pool was measured by bioassay. Positive animals were identified by ELISA detection of
258
PrPTSE on brain samples. Prion infected exosomal plasma pellets (EPPs) were prepared, treated with or
259
without proteinase K (PK) and analysed by western blot before and after sucrose gradient fractionation. IC,
260
intracerebral
Diagram of the main experimental procedure
261 262
Figure 2. Western blots of PrP in infected and control exosomal plasma pellets
263
a PrP positive immunoglobulin (Ig) bands were resistant to standard (50µg/ml; +) proteinase K (PK)
264
digestion (arrowheads) in both exosomal plasma pellets from control (cEPP) and infected (iEPP)
265
samples. The same conditions digested 37 kD PrP (arrow) of normal brain homogenate (NBH). After
266
stringent treatment with 100 µg/ml of PK (++), PrP-like bands were also seen, possibly due to
267
degradation (asterisk). EPPs of PrP knock-out (KO) mouse are positive to anti PrP SAF84 antibody. CD81
268
positive cEPP of both wild type (WT) and KO mice contain PrP positive Ig bands (arrowheads) that are
269
resistant to PK. No staining is observed without (W/O) the primary SAF84 antibody and in NBH. The
270
exosomal marker CD81 is shown in the lower panels. (n=3).
271
b Pre-absorption of anti-PrP SAF84 antibody with recombinant PrP (recPrP) (5:1) removes the recPrP
272
band, PrP bands (arrows) of NBH, and Ig bands of EPPs (arrowheads). No staining was observed in
273
absence of the primary SAF84 antibody. (n=3).
274
Numbers on the left indicate molecular weights in kD.
275 276 277
Figure 3
Silver staining and western blots of sucrose gradient fractions of infected and control exosomal plasma pellets
278
PAGE silver staining reveals that the bulk of plasma proteins are retained in the high-density fractions of
279
both exosomal plasma pellets from control (cEPP) and infected (iEPP) samples.
280
Western blots with SAF84 antibody show the presence of PrPC in fraction 6 (1.13 g/ml) of cEPPs (n=3),
281
which is abolished by the treatment with proteinase K (PK). PK resistant PrPTSE is shown in fraction 6 of
15 282
iEPPs. Western blots of sucrose fractions for exosomal markers (Tsg101, flotillin and CD81) show that
283
exosomes float in fractions 5 to 8. Absence of calnexin and nucleoporin indicates no cellular debris
284
contamination. nBH, normal hamster brain homogenate; sBH, scrapie-infected hamster brain homogenate.
Figure 1 Click here to download high resolution image
Figure 2 Click here to download high resolution image
Figure 3 Click here to download high resolution image
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary Figure Legends.pdf
Journal of General Virology Detection of exosomal prions in blood by immunochemistry techniques --Manuscript Draft-Manuscript Number:
JGV-D-14-00185R2
Full Title:
Detection of exosomal prions in blood by immunochemistry techniques
Short Title:
Exosomal prions in blood
Article Type:
Short Communication
Section/Category:
TSE Agents
Corresponding Author:
Maurizio Pocchiari Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità Roma, ITALY
First Author:
Francesca Properzi
Order of Authors:
Francesca Properzi Mariantonia Logozzi Hanin Abdel-Haq Cristina Federici Luana Lugini Tommaso Azzarito Ilaria Cristofaro Daniela di Sevo Elena Ferroni Franco Cardone Massimo Venditti Marisa Colone Emmanuel Comoy Valérie Durand Stefano Fais Maurizio Pocchiari
Abstract:
In most forms of prion diseases blood is infectious, but the detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of exosome on sucrose gradient was necessary for removing plasma immunoglobulins, which interfere with PrPTSE masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Properzi_text_revised_clean.docx
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Detection of exosomal prions in blood by immunochemistry techniques
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Running title: Exosomal prions in blood
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Francesca Properzi, Mariantonia Logozzi, Hanin Abdel-Haq, Cristina Federici, Luana Lugini, Tommaso
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Azzarito, Ilaria Cristofaro, Daniela di Sevo, Elena Ferroni, Franco Cardone, Massimo Venditti, Marisa Colone,
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Emmanuel Comoy, Valérie Durand, Stefano Fais and Maurizio Pocchiari
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Affiliations
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Francesca Properzi, Hanin Abdel-Haq, Ilaria Cristofaro, Daniela di Sevo, Elena Ferroni, Franco Cardone,
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Massimo Venditti, and Maurizio Pocchiari, Department of Cell Biology and Neurosciences, Istituto
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Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.
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Mariantonia Logozzi, Cristina Federici, Luana Lugini, Tommaso Azzarito, and Stefano Fais, Department of
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Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, 00161 Rome, Italy;
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Marisa Colone, Department of Technologies and Health, Istituto Superiore di Sanità, 00161 Rome, Italy
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Emmanuel Comoy and Valérie Durand, Institute of Emerging Diseases and Innovative Therapies, CEA,
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Fontenay-aux-Roses, France
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Corresponding author
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Maurizio Pocchiari
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Email: [email protected]
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Telephone: +39 06 49903203
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Fax: +39 06 49903805
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Word Count Summary: 93
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Tables:
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Figures:
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Suppl. Figures:
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Summary paragraph
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In most forms of prion diseases blood is infectious, but the detection by immunochemistry techniques of
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the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We
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developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the
34
finding that PrPTSE is associated with plasma exosomes. However, further purification of exosome on
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sucrose gradient was necessary for removing plasma immunoglobulins, which interfere with PrPTSE masking
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its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with
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exosomes.
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4 39
Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal neurodegenerative disorders
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affecting humans and animals caused by unique infectious particles (prions), which are mainly composed
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by misfolded forms of the cellular prion protein (PrPC). PrPC is constitutively expressed, but converts to the
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misfolded noxious structure (PrPTSE) either spontaneously or upon exposure to prions (Weissmann, 2004).
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Prions are efficiently transmitted via blood in most animal models of TSE diseases (Brown et al., 2001)
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(Houston et al., 2008) and in humans with variant and possibly sporadic CJD (Llewelyn et al., 2004) (Wroe
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et al., 2006) (Puopolo et al., 2011) (Douet et al., 2014). Prion infectivity in blood is associated with both
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cellular and non cellular components (Gregori et al., 2006) with estimated titres in plasma ranging from 1
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to 10 infectious unit/ml at disease onset (Brown et al., 2001) (Douet et al., 2014). Although plasma has
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been shown to carry infectivity, the identification of PrPTSE in infected animals and humans has been elusive
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and limited to the use of highly sensitive (Castilla et al., 2005) (Saá et al., 2006) (Pan et al., 2007) (Orrú et
50
al., 2011) or complex (Edgeworth et al., 2011) methodologies. Because blood components likely interfere
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with PrPTSE detection (Hartwell et al., 2005) (Abdel-Haq, 2014), we thought to bypass this difficulty by
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looking at plasma exosomes. Exosomes are nanovesicles that carry RNAs, proteins, lipids, other metabolites
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(Théry et al., 2009) (Fais et al., 2013), PrPC and, in prion-infected hosts, PrPTSE and infectivity (Fevrier et al.,
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2004) (Alais et al., 2008) (Coleman et al., 2012) (Saá et al., 2014).
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We first collected blood from intracerebrally 263K scrapie-infected hamsters (n=125) (Kimberlin and
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Walker, 1977) or from uninfected and healthy animals as controls. Plasma was separated from blood cells
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by centrifugation at 5,000 g for 30 minutes at room temperature. Single plasma fractions from infected
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hamsters were pooled to a final volume of 250 ml and used for the measurement of infectivity and
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purification of exosomes for the detection of PrPTSE by immunochemistry assays (Fig. 1). Bioassay was
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performed by intracerebral inoculation of 124 six weeks-old female Syrian hamsters with 50 µl of thawed
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plasma, under anesthesia with ketamine/xylazine (75 and 10 mg/kg respectively) mixture. Animals were
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housed in accordance to the national guidelines in level-3 animal care facilities (agreement numbers A 92-
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032-02 for animal care facilities, 92-189 for animal experimentation, CEA, France) and regularly monitored.
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Animals with clinical signs or with no disease up to 600-700 days were euthanized, the brain removed and
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tested for the presence of PrPTSE by the ELISA test as previously described (Barret et al., 2003). Briefly, PrPTSE
5 66
was extracted from brains by the Bio-Rad purification kit (USA), digested with proteinase K, and added into
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anti-PrP SAF53 (Bertin Pharma, France) monoclonal antibody (mAb)-coated ELISA wells. Bound PrPTSE was
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detected by incubation with the 11C6 (Bertin Pharma, France) anti-PrP mAb coupled with
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acetylcholinesterase, and revealed using the Ellman substrate at 414 nm optical wavelength.
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Eighteen animals resulted positive for scrapie (time to death, mean, 265 days; s.e.m., 23 days) with a
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corresponding infectivity titre of 3.1±0.7 infectious dose (ID) per ml (Supplementary Fig. 1), in line with
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previously published results on plasma samples (Gregori et al., 2006) (Brown et al., 2001). Infectivity titre
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(ID) was calculated by the limiting dilution method according to the Poisson distribution (Gregori et al.,
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2006).
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From the same pool of plasma, exosomes were isolated from 5 ml aliquots of control and infectious plasma
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pool by a previously published protocol (Caby et al., 2005). Briefly, exosome plasma pellets were prepared
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by serial centrifugations; the resulting pellets were washed in PBS, filtered through a 0.22 μm filter
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(Millipore, USA) and ultracentrifuged at 110,000g for 1 h. The exosome pellets were re-suspended in PBS,
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lysis buffer or in 2.5 M sucrose buffer, for further analyses as indicated in Fig. 1. Antibodies used for
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exosomal marker identification were the anti-Flotillin, anti-Tsg101, and anti-mouse CD81 (Santa Cruz,
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Germany). For PrP identification the primary antibodies used were SAF84 and biotinylated-SAF84 (Bertin
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Pharma, France), 3F4 (Millipore, Italy), and D18 (gift from R. A. Williamson, The Scripps, Florida). Western
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blot analyses were performed as described (Lee et al., 2000) and the consistency of these preparations
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confirmed by the detection of exosomal markers (Fig. 2a). Assuming a similar ratio between infectivity and
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PrPTSE molecules in blood and in brain tissue (about 0.1 to 1 pg PrPTSE/infectious unit) (Brown et al., 2001),
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we estimated that 25 µl of exosomal plasma pellets (EPPs), corresponding to 5 ml of the original plasma
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volume and 15.5 IDs, were suitable for detecting PrPTSE by standard western blot analyses (Lee et al., 2000).
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In EPPs from control and prion-infected samples, we found that anti-PrP antibodies recognised the Ig light
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chain (26 kD), and the IgG (53 kD) and IgM (65 kD) heavy chains as previously reported (Hartwell et al.,
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2005). These bands were resistant to an amount (50 µg/ml) of proteinase K (PK) that is largely sufficient to
91
remove the bulk of tissue proteins in brain samples (Fig. 2). The atypical bands were seen irrespectively of
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the anti-PrP primary antibody used (Supplementary Fig. 2) and were not detected in the absence of primary
6 93
antibodies (Fig. 2a). They were specific to plasma (Fig. 2a) and observed in EPPs of other species including
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humans (data not shown) and wild type mice (C57BL/10) (Fig. 2a and supplementary Fig. 2). They were also
95
reproduced in EPPs of PrP knock-out 129/Ola mice (gift from Dr Di Bari and Dr Agrimi, Istituto Superiore di
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Sanità, Rome, Italy) (Fig. 2a and supplementary Fig. 2) clearly indicating a cross-reaction of the primary
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antibodies with plasmatic PK-resistant material, unrelated to PrP epitopes. Finally, when primary anti-PrP
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antibodies were pre-absorbed with an excess of Syrian hamster 23-231 recombinant PrP (recPrP) (5:1),
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western blot membranes were negative, demonstrating that pre-absorbed primary antibodies are unable
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to bind PK-resistant Igs (IgRES). These data suggest that the levels of endogenous PrPC in plasma (in the
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range of pg/ml) is not sufficient to replace the non-specific binding of largely represented plasma proteins
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(in the range of mg/ml).
103
Stringent PK conditions (100 µg/ml), however, digested high molecular weight IgRES bands (Fig. 2a)
104
allowing the recognition of three bands of the same molecular weight of full length prion protein with a
105
glycoform ratio similar to that observed in hamster brain (Fig. 2a) (Meade-White et al., 2009). We then
106
further purified EPPs on a sucrose cushion starting from 15 ml of plasma to verify that these bands
107
effectively belong to full-length prion protein. Continuous sucrose gradient for the isolation and purification
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of exosomes was performed as published (Raposo et al., 1996), with some modifications. Briefly, purified
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exosomes were re-suspended in 2 ml of 2.5 M sucrose, 20 mM Hepes/NaOH, pH 7.2. A linear sucrose
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gradient (2.0-0.25 M sucrose) was layered on top of the exosomes suspension in a UltraClear™ tube and
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the sample was centrifuged at 110,000 g for 16 h at 4oC in a Beckman-Coulter (USA) SW41Ti rotor. Gradient
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fractions (12 x 1 ml) were collected from the top of the tube and washed with PBS. Fractions density was
113
evaluated using an Abbé Refractometer (Carl Zeiss, Germany).
114
Silver
115
http://imagej.nih.gov/ij/) showed that after sucrose gradient ultracentrifugation the bulk of the
116
immunoglobulins was retained at high-density fractions (1.64 to 1.70 g/ml), while exosomal fractions (1.11
117
to 1.13 g/ml) had significantly lower content of IgRES. On sucrose gradient purified EPPs, di-glycosylated
118
PrPC bands (37 kD) were detected at densities of 1.13 g/ml (Fig. 3) and disappeared after PK treatment (Fig.
119
3). This fraction was also positive on western blot to standard exosomal markers (Tsg101, Flotillin and
staining
after
PAGE
and
quantification
of
bands
using
ImageJ
(NIH,
available
at
7 120
CD81) and negative to ER and nucleus markers (calnexin and nucleoporin) indicating the purity of exosomal
121
preparation after sucrose gradient isolation (Fig. 3). As controls, ER and nucleus markers resulted positive in
122
the brain homogenate (Fig. 3).
123
In infectious EPPs, 37 kD PrP bands were observed in exosomal fractions at densities of 1.13 g/ml (Fig. 3).
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After treatment with PK, PrP-positive bands showed the characteristic shift in molecular weights of PrPTSE
125
(27-30 kD) with a glycoform ratio similar to that observed in 263K-infected brain homogenate (Fig. 3). The
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presence of PrPC on plasma exosomes was further ascertained by electron microscopy. Immunoelectron
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microscopy was performed on isolated sucrose gradient fractions of hamster plasma exosomes. A drop of
128
exosome suspension was placed onto a formvar-carbon-coated grid. After primary antibody (SAF84)
129
incubation exosomes were labelled with anti-rabbit IgG or anti-mouse IgG 5nm diameter-gold particle-
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conjugated antibody (Sigma-Aldrich, USA). Grids were negatively stained with uranyl acetate solution and
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examined with a Philips 208 transmission electron microscope (FEI Company, The Netherlands). In the 1.13
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g/ml fraction there was presence of nanovesicles, which after immunogold staining, were PrP-positive
133
particles on the surface of nanovesicles (~50 nm) (Supplementary Fig. 3a and 3b). On these samples,
134
nanovesicles of the same size were positive to the standard exosomal marker Rab5b and no PrP staining
135
was observed in fractions of lower and higher densities (Supplementary Fig. 3c).
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Finally, to further confirm the presence of prion in plasma exosomes we measured scrapie infectivity.
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However, for this experiment we used the mouse-adapted 139A scrapie strain (Dickinson, 1976), because
138
139A, but not 263K infectivity can be quickly and accurately measured by using the End Point Scrapie Cell
139
Assay (SCEPA) (Klöhn et al., 2003). Susceptible CAD5 cells (gift from Prof. Charles Weissmann, The Scripps,
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Florida, USA) were seeded in wells of 96-well plate and exposed to 12 or more aliquots of the 139A prion-
141
containing samples for 3 days, grown to confluence and split 1:3 three times and 1:10 twice. When the cells
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reached confluence, 25,000 cells were transferred to membranes of ELISpot plates and PrPTSE-containing
143
cells were identified as spots using the D18 antibody (Supplementary Fig. 4). A well is considered positive
144
when the following two criteria are fulfilled. Firstly, the number of positive spots must be higher then the
145
mean plus 5 times standard deviations of unseeded wells (background value). Secondly, prion propagation,
146
revealed by an increased number of positive spots after subsequent passages, must be evident. The ID was
8 147
calculated considering the number of negative wells over positive by using a Poisson distribution. A titred
148
brain homogenate is used as positive control to monitor variations caused by antibodies. Nanovesicles
149
were isolated from 3 ml of plasma of 139A-infected mice at terminal stage. We found 1 of 6 positive wells
150
for exosomal samples and 4 of 48 positive wells for the 1:5 dilution of plasma supernatant. Accordingly to
151
the Poisson distribution for rare events, the IDs contained in the processed volume (3 ml) were 1.1 for the
152
exosomal pellet and 4.3 for plasma supernatant. These findings confirm that in vivo prions are associated to
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nanovesicles similarly to what previously observed in vitro (Fevrier et al., 2004) (Alais et al., 2008) (Coleman
154
et al., 2012) (Fauré et al., 2006) (Vella et al., 2007) and that in plasma about 20% of total infectivity is
155
associated to exosomes leaving open the issue on how the remaining infectivity is distributed.
156 157
The most important finding of our work is the successful identification of PrP TSE in plasma of scrapie-
158
infected hamsters by standard western blot technique performed on exosomal fractions. This result
159
enforces previous data showing the presence of PrPTSE in blood exosomes by the protein misfolding cyclic
160
amplification (PMCA) method (Saá et al., 2014), in plasma by the PMCA (Castilla et al., 2005) and RT-QuiC
161
assays (Orrú et al., 2011) and in human vCJD patients by the solid-state binding matrix assay (Edgeworth et
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al., 2011), but adds the important novel information that PrPTSE, similarly to infectivity (Brown et al., 2001)
163
is detectable in blood without using amplification techniques. The detection of PrP TSE by the standard
164
western blot technique was only possible after reducing the interference of IgRES by exosomal pellet
165
fractionation on a sucrose gradient. IgRES compete for the primary antibody binding with endogenous PrP
166
epitopes crucially interfering with PrPTSE detection in blood. This result confirms that blood components
167
interfere with prion diagnosis (Hartwell et al., 2005) (Abdel-Haq, 2014) reducing the sensitivity and
168
specificity of the various tested assays. Moreover, these findings might explain the discrepancy between
169
the excellent performances of tests accomplished on spiked blood and their failure on endogenously
170
infected blood that is likely related to different interaction with plasma Igs between exogenous and
171
endogenous PrPTSE.
172 173
9 174
Acknowledgments
175
We thank Dr Anna Ladogana for critical revision of the manuscript, Giuseppe Loreto for the preparations of
176
the figures, Dr Agnese Molinari for support with EM, Nick Verity for supplying the SCEPA samples, and Dr
177
Alessandra Garozzo for editorial assistance.
178
This work was partially supported by the Istituto Superiore di Sanità, Rome, Italy and by the Alliance
179
Biosecure Project “Plasmasecure”.
180 181
10 182
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Figure Legends
256
Figure 1
257
Infectivity of plasma pool was measured by bioassay. Positive animals were identified by ELISA detection of
258
PrPTSE on brain samples. Prion infected exosomal plasma pellets (EPPs) were prepared, treated with or
259
without proteinase K (PK) and analysed by western blot before and after sucrose gradient fractionation. IC,
260
intracerebral
Diagram of the main experimental procedure
261 262
Figure 2. Western blots of PrP in infected and control exosomal plasma pellets
263
a PrP positive immunoglobulin (Ig) bands were resistant to standard (50µg/ml; +) proteinase K (PK)
264
digestion (arrowheads) in both exosomal plasma pellets from control (cEPP) and infected (iEPP)
265
samples. The same conditions digested 37 kD PrP (arrow) of normal brain homogenate (NBH). After
266
stringent treatment with 100 µg/ml of PK (++), PrP-like bands were also seen, possibly due to
267
degradation (asterisk). EPPs of PrP knock-out (KO) mouse are positive to anti PrP SAF84 antibody. CD81
268
positive cEPP of both wild type (WT) and KO mice contain PrP positive Ig bands (arrowheads) that are
269
resistant to PK. No staining is observed without (W/O) the primary SAF84 antibody and in NBH. The
270
exosomal marker CD81 is shown in the lower panels. (n=3).
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b Pre-absorption of anti-PrP SAF84 antibody with recombinant PrP (recPrP) (5:1) removes the recPrP
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band, PrP bands (arrows) of NBH, and Ig bands of EPPs (arrowheads). No staining was observed in
273
absence of the primary SAF84 antibody. (n=3).
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Numbers on the left indicate molecular weights in kD.
275 276 277
Figure 3
Silver staining and western blots of sucrose gradient fractions of infected and control exosomal plasma pellets
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PAGE silver staining reveals that the bulk of plasma proteins are retained in the high-density fractions of
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both exosomal plasma pellets from control (cEPP) and infected (iEPP) samples.
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Western blots with SAF84 antibody show the presence of PrPC in fraction 6 (1.13 g/ml) of cEPPs (n=3),
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which is abolished by the treatment with proteinase K (PK). PK resistant PrPTSE is shown in fraction 6 of
15 282
iEPPs. Western blots of sucrose fractions for exosomal markers (Tsg101, flotillin and CD81) show that
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exosomes float in fractions 5 to 8. Absence of calnexin and nucleoporin indicates no cellular debris
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contamination. nBH, normal hamster brain homogenate; sBH, scrapie-infected hamster brain homogenate.
Figure 1 Click here to download high resolution image
Figure 2 Click here to download high resolution image
Figure 3 Click here to download high resolution image
Supplementary Material Files Click here to download Supplementary Material Files: Supplementary Figure Legends.pdf
