Written informed consent for participation in research and all procedures for sample collection and experimental studies were in accordance with the 1964 Declaration of Helsinki and its later amendments and were approved by the Ethical Committee of “Fondazione IRCCS Istituto Neurologico Carlo Besta” (Milan, Italy). Tg(MHu2M)FVB-B5378 mice were housed in individually ventilated cages (2–5 mice per cage), daily fed, and provided with water. Lighting was on an automatic 12 h basis. Regular care was periodically performed for assessment of animal health. The animal facility is licensed and inspected by the Italian Ministry of Health. Current animal husbandry and housing practices comply with the Council of Europe Convention ETS123 (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes; Strasbourg, 18.03.1986), Italian Legislative Decree 116/92 (Gazzetta Ufficiale della Repubblica Italiana, 18 February 1992), and with the 86/609/EEC (Council Directive of 24 November 1986 on the approximation of laws, regulations, and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes). The animal study, including its Ethics aspects, was approved by the Italian Ministry of Health (Permit Number: 258/2018-PR), and all efforts were made to minimize animals suffering.

Murine genomic DNA was extracted from ear biopsies and analyzed by PCR using specific primers (5′-GAACTG AACCATTTCAACCGAG-3′ and 5′-AGAGCTACAGGTGGA TAACC-3′). Denaturation at 95°C for 5 min was followed by 35 cycles of amplification (95°C for 1 min, 58°C for 1 min, 72°C for 1 min). PCR products were resolved by agarose gel electrophoresis and analyzed for the presence of the transgene. Human DNA was extracted from peripheral blood mononuclear cells (PBMC). The coding sequence of PRNP gene was amplified by PCR using two pairs of primers: forward 5′-CAGAGAAGTACAGGGTGGCA-3′/reverse 5′-AATGTATGA TGGGCCTGCTCAT-3′ and forward 5′-CAACATGAAGCAC ATGGCTGGT-3′/reverse 5′-TAAAAGGGCTGCAGGTGGAT AC-3′. The amplified fragments were sequenced using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and analyzed on an ABI 3130xl Genetic Analyzer (Applied Biosystem).

A total of 65 OM samples were included in the study (see Table 1). In particular, 27 were collected from sCJD patients (MM = 13, MV = 8 and VV = 6), 2 from genetic CJD (gCJD) patients harboring the E200K mutation and 36 from patients with other neurodegenerative/neurological disorders (OND), including Alzheimer’s disease (AD = 3) (McKhann et al., 2011), Parkinson’s disease (PD = 7) (Postuma et al., 2015), frontotemporal dementia (FTD = 7) (Rascovsky et al., 2011), multiple system atrophy (MSA = 4) (Gilman et al., 2008), progressive supranuclear palsy (PSP = 7) (Höglinger et al., 2017), corticobasal degeneration (CBD = 6) (Alexander et al., 2014) and multiple sclerosis (MS = 2) (Table 1). OM samples were collected between the septum and the middle turbinate in the vault of the nose by brushing with a cotton swab, as previously described (De Luca et al., 2019). Cotton swabs were then immersed in saline solution and subjected to vigorous shaking to collect OM cells. Samples were then stored at −80°C until analyses. A total of 28 brains were included in the study. In particular, frozen samples of frontal cortex (gyrus cinguli) were collected from 26 sCJD patients (MM1 = 7, MV1 = 3, VV1 = 1, MM2-cortical = 3, MM2-thalamic = 2, MV2 = 6, VV2 = 4) and 2 non-CJD patients (AD = 1_129MM and FTD = 1_129MV). Sixteen of the sCJD brains (MM1 = 6, MV2 = 6, and VV2 = 4) belonged to patients who underwent OM collection (see Table 2 for details). The demografic data, genetic, instrumental and laboratory findings of all sCJD patients who underwent OM collection are reported in Table 3. The brains of the AD and FTD patients who donated OM samples were not collected.

Frozen brain samples (sCJD, AD and FTD) were homogenized at 10% (weight/volume, w/v) in lysis buffer (100 mM sodium chloride, 10 mM ethylenediaminetetraacetic acid tetrasodium salt (EDTA), 0.5% Non-idet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH 7.4). Brain homogenates (BHs) were then centrifuged at 4°C, 800 × g, for 1 min to remove cellular debris. The supernatant was then subjected to different analyses. In particular, 10 μL was treated with PK and subjected to Western blot (Wb) analysis to verify the presence of PrP^res and determine its biochemical properties (glycoform ratio and typing). Fifty μL was used to perform PK resistance analysis (as detailed in the section “Proteinase-K Resistant Profile of Brain Homogenates and PMCA Generated Products”). Twenty μL of selected BHs was used to perform Peptide-N-Glycosidase F (PNGase F) experiments (as detailed in the section “PNGase F Treatment of Human Brain Homogenates”). Each BH was diluted at 10^–4 in phosphate buffer saline (PBS) (volume/volume, v/v) and 2 μL of this dilution was subjected to RT-QuIC analysis (as detailed in the section “RT-QuIC Analysis of Brain Homogenates and Olfactory Mucosa”). Finally, each BH was diluted from 10^–3 to 10^–12 in PBS (v/v) and every dilution was subjected to PMCA analysis (as detailed in the section “PMCA Analysis of Brain Homogenates and Olfactory Mucosa”).

Olfactory mucosa samples were thawed and centrifuged at 4°C, 800 × g, for 20 min. The saline solution was removed and approximately 2 μg of the pellet was collected using an inoculating loop, and dissolved in 25 μL of PBS (pH 7.4, Sigma). Ten μL of this solution was used to perform PMCA analysis (as detailed in the section “PMCA Analysis of Brain Homogenates and Olfactory Mucosa”). Two μL was further diluted into 18 μL of PBS and 2 μL of this latter dilution was used to perform RT-QuIC analysis (as detailed in the section “RT-QuIC Analysis of Brain Homogenates and Olfactory Mucosa”).

Full-length human PrP with methionine at position 129 (recHuPrP_23–231) and truncated Syrian hamster PrP (recHaPrP_90–231) constructs were expressed in Escherichia coli BL21 (DE3) cells (Stratagene). One hundred mL of overnight culture was inoculated into Luria-Bertani (LB) medium complemented with 100 μg/mL ampicillin. At 0.6 OD600 the expression of the constructs was induced with 0.8 mM of isopropyl b-D galactopyranoside (IPTG). Cells were grown at 30°C for 12 h and then lysed in 25 mM Tris–HCl, 5 mM EDTA, 0.8% Triton X-100, pH 8.0 with fresh added 1 mM phenylmethylsulfonyl fluoride (PMSF) using PandaPLUS 2000. Inclusion bodies containing the recombinant proteins were washed several times in bi-distilled water and then dissolved in 8 M guanidine hydrochloride (GdnHCl) before loading onto a pre-equilibrated HiLoad 26/60 Superdex 200 pg column (Cytiva) and eluted in 5 M GdnHCl, 25 mM Tris–HCl, 5 mM EDTA, pH 8.0 at a flow rate of 2 mL/min. Protein refolding was performed by dialysis against 20 mM sodium acetate, pH 5.5 using Spectrapor membrane. Purified proteins were analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions, dialyzed against phosphate buffer, pH 5.8 and stored at −80°C. All salts used were from Sigma-Aldrich. RecHaPrP_90–231 was used as a reaction substrate for RT-QuIC analyses, while recHuPrP_23–231 was used for quantitative PMCA.

RecHaPrP_90–231 was thawed and filtered through a 100 kDa Nanosep centrifugal device (Pall Corporation). RT-QuIC reaction mix was composed of 10 mM PBS (pH 7.4), 150 mM NaCl, 0.13 mg/ml recHaPrP_90–231, 1 mM EDTA, 0.002% SDS and 10 μM thioflavin T (ThT). Ninety−eight μL of the reaction mix was placed in a black 96-well optical flat bottom plate (Thermo Scientific) and supplemented with 2 μl of OM or BH (prepared as previously described). Each sample has been analyzed in quadruplicate. The plate was sealed with a sealing film (Thermo Scientific) and underwent intermittent cycles of shaking (1 min at 600 rpm, double orbital) and incubation (1 min) at 55°C using the fluorescence microplate reader OPTIMA (BMG Labtech). ThT fluorescence was measured every 30 min (wave-lengths: excitation 450 ± 10 nm; emission 480 ± 10 nm). A sample was considered positive if the fluorescence value of at least 2 out of 4 replicates was higher than 10,000 arbitrary units (AU) before the threshold of time set at 60 h, as described (Franceschini et al., 2017).

The brains of Tg(MHu2M)FVB-B5378 mice (Telling et al., 1994), from now on referred to as TgHuMM mice, were homogenized at 10% (w/v) in conversion buffer (PBS 1X containing 150 mM sodium chloride and 1% Triton X-100) supplemented with cOmplete Mini EDTA-free protease inhibitor cocktail (Roche) and used as a PMCA reaction substrate. To increase PMCA efficiency, 0.135 M sodium tripolyphosphate, 6 mM EDTA, 100 μg/mL heparin, 0.05% digitonin (Sigma), and 3 Teflon beads were used to supplement the substrate.

Ten μL of each dilution of BH or OM sample (prepared as previously described) was added to 90 μL of PMCA substrate, transferred into 0.2 mL PCR tubes and subjected to amplification using a Qsonica Q700 sonicator. PMCA consisted of intermittent cycles of incubation (29 min and 20 s) and sonication (40 s set at 260–280 W). After 48 h of reaction (considered as a round of amplification), 10 μL of the amplified material was added to 90 μL of freshly prepared PMCA substrate and an additional round of amplification was performed. In total, each sample (OM or BH) was subjected to 6 (or in specific cases to 7) PMCA rounds. To avoid any contamination, PMCA substrates were prepared under rigorous prion-free conditions and the sonicator horn was periodically decontaminated with 4 M Gdn-HCl (overnight). To monitor the contamination and the potential de novo generation of prions, appropriate negative controls were included in each PMCA round. All the analyses have been repeated three times by three different operators to check the reproducibility of the results. Amplified products obtained from BH were named BH_PMCA while those obtained from OM were named OM_PMCA.

Ten μL of (i) BHs (sCJD, AD and FTD), (ii) BH_PMCA and (iii) OM_PMCA were treated with 50 μg/mL of proteinase K (PK, Invitrogen) for 1 h at 37°C under shaking (550 rpm) before Wb analysis and immunoblotting with 6D11 antibody (1:5,000, epitopes 93–109, Covance). Ten μL of BHs were digested with 400 μg/mL of PK for Wb analysis and immunoblotting with 12B2 antibody (1:8,000, epitopes 89–93).

The PK resistant profile of the samples marked with red and/or black asterisks in Table 2 has been assessed. In particular, the black asterisk refers to the analysis of raw BHs (22 samples: MM1 = 5, MM2C = 3, MM2T = 2, MV1 = 3, MV2 = 4, VV1 = 1, VV2 = 4) while the red asterisk refers to the analysis of BH_PMCA (20 samples: MM1 = 5, MM2C = 2, MM2T = 2, MV1 = 3, MV2 = 4, VV2 = 4) and OM_PMCA [21 samples: MM = 9 (4 MM1, 5 unknown PrP^res typing), MV = 7 (5 MV2, 2 unknown PrP^res typing), VV = 5 (3 VV2, 2 unknown PrP^res typing)]. Each sample was treated with five increasing concentrations of PK (50, 250, 500, 1,000, and 1,500 μg/mL) and incubated for 1 h at 37°C under shaking (500 rpm). The enzymatic activity was stopped by boiling the sample for 10 min in loading buffer (Bolt™ LDS Sample Buffer and Dithiothreitol (DTT), Thermo Scientific) before Wb analyses. Densitometric quantification of PrP^res bands was performed as described in the section “Statistical Analysis.”

Proteinase K digested samples were loaded into 12% BisTris plus gels (Thermo Scientific) and subjected to electrophoresis analysis under denaturing conditions (SDS-PAGE). Samples that did not require PK treatment (e.g. recHuPrP_23–231) were immediately supplemented with loading buffer (Bolt™ LDS Sample Buffer 4X and DTT 10X, ThermoScientific) and boiled for 10 min before SDS-PAGE. Proteins were then transferred into polyvinylidene difluoride membranes (PVDF, Millipore) and incubated with non-fat dry milk (Santa Cruz) for 1 h at room temperature. PVDF membrane was probed using the monoclonal anti-PrP antibody 6D11 and then incubated with Fab fragment anti-mouse IgG conjugated with horseradish peroxidase (HRP) (GE). Blots were developed with a chemiluminescent system (ECL Prime, GE Healthcare Amersham). When required, densitometric quantification of PrP^res bands was performed as described in the section “Statistical Analysis.”

Twenty μL of selected BHs (3_BH, 16_BH, 19_BH, 25_BH, 26_BH, see below for details) was digested with 100 μg/mL of PK for 1 h at 37°C. PNGase F treatment was performed according to the manufacturer’s instructions (New England Biolabs PNGase F P0704S). Briefly, after digestion samples were supplemented with 4 μL di 10X glycoprotein denaturing buffer and boiled for 10 min. Then, samples were supplemented with 4 μL of reaction buffer (10X), 4 μL of NP-40 (10%), 4 μL of PNGase F and 4 μL of PBS and incubated overnight at 37°C. After incubation, 20 μL of LDS-PAGE loading buffer (Sample buffer 4X and DTT 10X, Thermo Scientific) was added to the samples that were boiled for 10 min. Finally, four serial dilutions (1:2) of each sample were prepared, loaded into 12% BisTris plus gels (Thermo Scientific) and subjected to Wb analysis.

Quantitative PMCA (qPMCA) was performed as previously described (Chen et al., 2010; Moda et al., 2014). In particular, we have selected 5 patients (3, 16, 19, 25 and 26) with known sCJD subtypes (MM1 = 1, MV2 = 2, VV2 = 2) in which prions were efficiently amplified from both the OM and the corresponding brain samples (see Table 2 for details). To this aim, known amounts of recHuPrP_23–231 (8, 6, 4, 2, and 1 ng) were analyzed by SDS-PAGE together with the dilutions of the PNGase treated BHs, to estimate their specific PrP^res content. Samples were immunoblotted with the 6D11 antibody and the intensities of each recHuPrP_23–231 band (representing known amounts of protein) were compared with those of the PrP^res present in each BH dilution. In this way, we have estimated the concentration of PrP^res in every sCJD-BH. Densitometric analysis of all resulting PrP bands was performed as described in the section “Statistical Analysis.”

Densitometric analysis of Western blot bands were carried out using ImageJ software (1.48v). Graphic representations of densitometric analysis were performed using the Prism software (GraphPad v7.0.5). PK resistance profiles of PrP^res derived from BH, BH_PMCA and OM_PMCA were analyzed through repeated analysis of variance (ANOVA). Associations between variables were investigated through t-test or Mann–Whitney test and Fisher exact test, as appropriate.

RT-QuIC analysis of the OM showed that all samples collected from sporadic and genetic CJD patients (n = 29), regardless of the polymorphism at PRNP129, induced an efficient seeding activity for recHaPrP_90–231 before 35 h. The reaction was stopped at 60 h and none of the OM collected from patients with other neurodegenerative/neurological disorders (OND) induced any seeding activity (Supplementary Figure 1A). We have then subjected to RT-QuIC analysis the brains of the 16 sCJD patients who underwent OM collection during life. Also in this case, regardless of the polymorphism at PRNP129, all sCJD brains induced an efficient seeding activity while AD and FTD brains did not (Supplementary Figure 1B).

The brain homogenates (BHs) of 26 sCJD patients were analyzed by Western blot (Wb) to confirm prion disease diagnosis and determine the biochemical properties of PrP^res. According to the polymorphism at PRNP129 and the PrP^res properties, samples were classified in the following groups: (i) MM1 = 7, (ii) MV1 = 3, (iii) VV1 = 1, (iv) MM2-cortical = 3 or (v) MM2-thalamic = 2 (differentiated by histopathological analysis), (vi) MV2 = 6 and (vii) VV2 = 4 (Supplementary Figures 2A–E). Sixteen of these brains belonged to patients whom the OM has been collected from: MM1 = 6, MV2 = 6, and VV2 = 4 (see Table 2 for details). The BHs of a patient with AD (MM) and a patient with FTD (MV) were included as negative controls and no PrP^res was detected (Supplementary Figure 2E). The same samples have been immunoblotted with the 12B2 antibody, which recognizes type 1 PrP^res, and in 6 MV2 and 4 VV2 samples we could detect the co-occurrence of type 1 and type 2 PrP^res (Supplementary Figures 2F–J).

To test the amplification efficiency of sCJD prions, we have subjected all 26 sCJD BHs to PMCA analysis (see Table 2). Regarding the MM1 subtype, prions were detected in 5/7 BHs at the 6^th round. In particular, in three BHs (7, 14 and 30) PrP^res was amplified with high efficiency (dilutions 10^–8–10^–12) while in two BHs (3 and 12) the amplification was less efficient and PrP^res was detected only in lower dilutions (10^–3–10^–5). No PrP^res was detected in two BHs (11 and 13) even after 6 rounds of amplification (Figure 1A). Notably, except for 7_BH, all amplified PrP^res switched from type 1 to type 2 and densitometric analysis revealed the presence of higher levels of the di-glycosylated species (Supplementary Figure 3A). Contrarily, in 7_BH the amplified PrP^res did not switch typing and the di-glycosylated and mono-glycosylated PrP bands were equally expressed (Supplementary Figure 3A). In the case of cortical and thalamic MM2 subtypes (MM2C and MM2T, respectively), PrP^res was amplified with low efficiency (dilutions 10^–3–10^–6) in 2/3 MM2C BHs (31 and 33) (Figure 1B) and all MM2T (38 and 39) (Figure 1C). No PrP^res was detected in 32_BH (MM2C) even after 6 rounds of amplification. Prions amplified from these BHs maintained type 2 profile but their glycoform ratios differed from those of the original strains and a predominant di-glycosylated band was observed (Supplementary Figure 3A). Regarding the MV subtypes, we could efficiently amplify PrP^res (dilutions 10^–6–10^–12) in all MV1 BHs (34, 35 and 36) (Figure 1D) and 5/6 MV2 (16, 19, 20, 21 and 22) (Figure 1E). No PrP^res was detected in 23_BH (MV2) even after 6 rounds of amplification. In both subtypes, the amplification led to the formation of type 1 PrP^res with the di-glycoyslated species predominant over the others (Supplementary Figure 3B). Thus, prions amplified in MV1 samples maintained the typing of the original inocula while those amplified in MV2 switched from 2 to 1. Finally, we efficiently amplified prions (dilutions 10^–6–10^–12) in all VV2 BHs (25, 26, 27 and 29) that showed type 1 PrP^res but in two of them (27 and 29) the di-glycoyslated species prevailed while in the other two (25 and 26) the di- and mono-glycosylated bands were equally represented (Supplementary Figure 3C). Hence, also in this case, amplified PrP^res did not maintain the typing of the original strain (Figure 1G). No PrP^res was amplified in the only available VV1 sample (37_BH) even after 6 rounds of amplification (Figure 1F). As expected, no PrP^res was amplified from the brains of AD or FTD patients (Figure 1H). Regardless of the co-occurrence of type 1 and type 2 PrP^res in all MV2 and VV2 BHs, the amplified products acquired similar biochemical properties thus suggesting that the PMCA was able to generate a distinctive prion isolate (Polymenidou et al., 2005; Notari et al., 2007; Cali et al., 2020). Interestingly, these particular switches of PrP^res typing after PMCA analysis of sCJD brain homogenates were recently described by the group of Bougard (Bélondrade et al., 2021).

To test whether our PMCA protocol could also amplify prions in the OM of sCJD patients, we have analyzed 65 samples collected from 27 sCJD, 2 gCJD, and 36 OND patients (see Table 1 for details). From the whole group of MM samples (n = 14), we could amplify PrP^res in 10 OM (71.4%), including 4 MM1, 1 E200K and 5 samples with unknown PrP^res typing (MMunk). Two MM1 and 2 MMunk remained negative (Figures 2A,B). The majority of the PrP^res amplified in this group of OM were of type 1 and characterized by an equal representation of the di- and mono-glycosylated species (1, 4, 8, 10, 11, 12 and 14). In contrast, three of them (3, 5 and 6) were of type 2 with a prevalence of the di-glycosylated species (Figures 2A–C and Supplementary Figure 3A). Considering only the MM1 samples, we were able to amplify prions in 4/6 OM (67%). Interestingly, 3 OM that generated type 1 PrP^res (11, 12 and 14) had the corresponding BHs that amplified type 2 PrP^res, except for 11_BH that remained negative (Figure 1). The remaining OM (number 3) and the corresponding BH (number 3 in Figure 1) amplified type 2 PrP^res. Therefore, the biochemical properties of MM prions (in terms of typing or glycoform ratio) were not faithfully maintained after the amplification. Surprisingly, even the PrP^res amplified from BH and OM of the same sCJD patient often possessed distinct biochemical features (e.g., in patient 12 type 1 PrP^res was amplified from BH while type 2 PrP^res was amplified from the corresponding OM) (Figures 1A, 2A and Supplementary Figure 3A). From the whole group of MV samples (n = 9), we were able to amplify PrP^res in 8 OM (88.8%), including 5 MV2, 1 E200K and 2 samples with unknown PrP^res typing (MVunk), while 1 MV2 remained negative (Figures 2A,B). All amplified products in this group of OM (15, 16, 17, 18, 19, 20, 22 and 23) were characterized by type 1 PrP^res with an equal representation of the di- and mono-glycosylated species (Figures 2A–C and Supplementary Figure 3B). Considering only the MV2 samples, we were able to amplify prions in 5/6 OM (83.3%). Interestingly, their corresponding BHs amplified type 1 PrP^res but with a distinct glycoform ratio that was characterized by predominant di-glycosylated bands, instead (Supplementary Figure 3B). Finally, from the whole group of VV samples (n = 6), we were able to amplify PrP^res in 5 OM (83.3%), including 3 VV2 and 2 samples with unknown PrP^res typing (VVunk), while 1 VV2 remained negative (Figures 2A,B). All amplified products in this last group of OM (24, 25, 26, 27 and 28) showed type 1 PrP^res with an equal representation of the di- and mono-glycosylated species (Figures 2A–C and Supplementary Figure 3C). Considering only the VV2 samples, we were able to amplify prions in 3/4 OM (75%). Their corresponding BHs also amplified type 1 PrP^res but their glycoform ratio was characterized by a higher representation of the di-glycosylated band (27_BH and 29_BH) or an equal representation of di- and mono-glycosylated species (25_BH and 26_BH) (Supplementary Figure 3C). We have then evaluated at which PMCA round the OM samples showed detectable PrP^res and found that: 3 MM (3, 5 and 8) and 1 VV (number 26) showed PrP^res at the 3^rd PMCA round; 4 MM (6, 10, 11 and 12), 2 MV (16 and 20), and 3 VV (24, 25 and 28) showed PrP^res at the 5^th PMCA round; while 3 MM (1, 4 and 14), 6 MV (15, 17, 18, 19, 22 and 23), and 1 VV (number 27) showed PrP^res at the 6^th PMCA round (Figure 2B). No PrP^res was detected in the 36 OM samples of OND patients collected at the 6^th round (Figure 2A and Supplementary Figure 4). The biochemical properties of PrP^res detected in BH, BH_PMCA and OM_PMCA are summarized in Supplementary Table 1. All samples were analyzed at least three time by three different operators and analogous results were always obtained, thus confirming the reproducibility of the analytical procedures and the reliability of our findings.

Considering that the PrP^res amplified from BH and OM did not retain the specific features of the original prions and that, in most of the cases, they also differed between samples belonging to the same patients, we decided to test whether and to what extent the PMCA could have altered the prion properties during the amplification.

Initially, we have evaluated if the original prions might have undergone processes of selection and adaptation during the amplification, finally leading to the onset of different isolates. To this aim, we have selected 3 MM1 patients (number 3, 7 and 12), 1 MV2 patient (number 16), and 1 VV2 patient (number 26) whose PMCA results obtained from BH and OM were the most representative for their specific sCJD subgroup (Figure 3). In particular, we have observed that (i) BH and OM of patient 3 generated type 2 PrP^res with a prevalent di-glycosylated species (Figure 3A and Supplementary Figure 3A); (ii) BH and OM of patient 12 gave rise to type 2 and type 1 PrP^res, respectively, with glycoform ratios that differed from each other (Figure 3B and Supplementary Figure 3A); (iii) BH of patient 7 generated type 1 PrP^res with an equal representation of the di- and mono-glycosylated species while the OM remained negative (Figure 3C and Supplementary Figure 3A); (iv) BH and OM of patient 16 generated type 1 PrP^res with a prevalent di-glycosylated band (Figure 3D and Supplementary Figure 3B) and (v) BH and OM of patient 26 generated type 1 PrP^res with the di- and mono-glycosylated isoforms similarly represented (Figure 3E and Supplementary Figure 3C). Unfortunately, we could not perform similar evaluations in MM2C, MM2T, MV1 and VV1 patients because their OM were not available. Figure 3 shows that from the round at which the amplified PrP^res could be detected by Wb, significant changes in their biochemical profiles were not observed. However, we do not know whether an adaptation process might have occurred in the first rounds of PMCA where the amplified prions could not yet be visualized.

We have then evaluated whether the sensitivity to PK digestion of each sCJD prion was lost after the amplification or retained instead. First of all, we have treated all BHs and their products of amplification (BH_PMCA) collected at the 6^th PMCA round with increasing concentrations of PK and found that, in the case of MM2C, MM2T and VV2 the amplified PrP^res were significantly more sensitive to digestion than the corresponding original strains [repeated measure analysis of variance (ANOVA): p = 0.0001, p = 0.0103, p < 0.0001, respectively]. In the case of MM1, MV1 and MV2, these differences were still present but they were not statistically significant (p = 0.0805, p = 0.0366 and p = 0.1200, respectively) (Supplementary Figure 5). Notably, all PrP^res amplified from BHs showed similar PK resistance profile (p = 0.4137) (Figure 4A).

We have then treated the OM amplified samples (OM_PMCA) with the same concentrations of PK and found that, if combined together, the amplified PrP^res were significantly less resistant to PK with respect to those of BH_PMCA (p < 0.0001). Also in this case, the PK resistance profiles of OM_PMCA prions were comparable between each other (p = 0.9616) (Figure 4B). We have then performed additional analyses by considering only samples (BH, BH_PMCA and OM_PMCA) collected from 9 autopsied cases: 3 MM1 (number 3, 12 and 14), 3 MV2 (number 16, 19 and 20) and 3 VV2 (number 25, 26 and 27). The results of this analysis are reported in Figure 4 and show that the PrP^res present in BH, BH_PMCA and OM_PMCA of VV2 possessed significantly different PK resistance profiles between each other (p < 0.0001) (Figure 4E). In the case of MM1 patients, statistically significant differences in the PK resistance profiles were observed between BH and BH_PMCA (p < 0.0001) or BH and OM_PMCA (p < 0.0001) but not between BH_PMCA and OM_PMCA (p = 0.6004). Regarding MV2 patients, we have observed statistically significant differences between the PK resistance profiles of BH and OM_PMCA (p = 0.0018) but not between BH and BH_PMCA (p = 0.1183) or BH_PMCA and OM_PMCA (p = 0.2492) (Figures 4C,D). Taken together these data indicate that although PMCA could amplify, with variable efficiency, PrP^Sc from the majority of BH and OM samples, the features of the original sCJD prions were altered thus hampering the possibility of their recognition.

Through quantitative PMCA, we were able to estimate for the first time the concentration of prions in the OM samples of sCJD patients. Particularly, we have included one MM1 patient (patient 3), two MV2 patients (patient 16 and patient 19) and two VV2 patients (patient 25 and patient 26). Quantitative PMCA was performed following the protocol previously published by some of the authors of this manuscript (Chen et al., 2010; Moda et al., 2014; Redaelli et al., 2017). A highly accurate estimation of OM prions was made possible thanks to the availability of the corresponding brain samples that were used to calibrate the PMCA reactions. At the beginning, we have determined the PrP^res concentration in the brain of each sCJD patient. Brains were digested with PK treated with PNGase and analyzed by Wb (Figure 5B) along with known concentrations of human recombinant PrP (Figure 5A). In this way, we calculated that the PrP^res concentrations in 10 μL of the brains were as follow: patient 3 (3_BH): 8.42 ng (Figure 5C); patient 16 (16_BH): 5.69 ng (Figure 5D); patient 19 (19_BH): 10.96 ng (Figure 5D); patient 25 (25_BH): 14.72 ng (Figure 5E); and patient 26 (26_BH): 18.62 ng (Figure 5E). Then, we have performed serial dilutions of these BHs that were subjected to PMCA analysis together with the corresponding OM samples. We have observed that after 3 rounds, prions were detected in the OM of patient 3 (3_OM). The last PrP^res dilution amplified from BH at the 3^rd round of this patient corresponded to 8.4 × 10^–11 g, thus, by postulating that prions contained in BH and OM possess the same seeding activity by PMCA, this might be the approximate concentration of PrP^res present in 0.8 μg of OM (Figure 5C). Considering the rapidity of PrP^res amplification in this sample, we have verified whether it could contain a quantity of prions detectable by Wb even without amplification but we did not see any PK resistant signal (Supplementary Figure 6). The OM of patient 16 (16_OM) showed PrP^res at the 5^th round while that of patient 19 (19_OM) at the 6^th round which, according to the amplification of their brain dilutions, corresponded to 5.69 × 10^–20g (Figure 5D) and 1.096 × 10^–21g (Figure 5D) of protein, both detectable in 0.8 μg of sample. Finally, the OM of patient 25 (OM_25) showed PrP^res at the 5^th round while that of patient 26 (OM_26) at the 3^rd round. In this case, according to the last dilutions amplified from the corresponding BHs, we could estimate that PrP^res concentrations in 0.8 μg of these samples are approximately 1.47 × 10^–21g (Figure 5E) and 1.86 × 10^–13g (Figure 5E), respectively (Supplementary Table 2).