In situ photodegradation of incorporated polyanion does not alter prion infectivity

Single-stranded polyanions ≥40 bases in length facilitate the formation of hamster scrapie prions in vitro, and polyanions co-localize with PrP(Sc) aggregates in vivo. To test the hypothesis that intact polyanionic molecules might serve as a structural backbone essential for maintaining the infectious conformation(s) of PrP(Sc), we produced synthetic prions using a photocleavable, 100-base oligonucleotide (PC-oligo). In serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified PrP(C) substrate, PC-oligo was incorporated into physical complexes with PrP(Sc) molecules that were resistant to benzonase digestion. Exposure of these nuclease-resistant prion complexes to long wave ultraviolet light (315 nm) induced degradation of PC-oligo into 5 base fragments. Light-induced photolysis of incorporated PC-oligo did not alter the infectivity of in vitro-generated prions, as determined by bioassay in hamsters and brain homogenate sPMCA assays. Neuropathological analysis also revealed no significant differences in the neurotropism of prions containing intact versus degraded PC-oligo. These results show that polyanions >5 bases in length are not required for maintaining the infectious properties of in vitro-generated scrapie prions, and indicate that such properties are maintained either by short polyanion remnants, other co-purified cofactors, or by PrP(Sc) molecules alone.     

A protease-resistant prion protein isoform is present in urine of animals and humans affected with prion diseases.

Prion protein (PrP)(Sc), the only known component of the prion, is present mostly in the brains of animals and humans affected with prion diseases. We now show that a protease-resistant PrP isoform can also be detected in the urine of hamsters, cattle, and humans suffering from transmissible spongiform encephalopathies. Most important, this PrP isoform (UPrP(Sc)) was also found in the urine of hamsters inoculated with prions long before the appearance of clinical signs. Interestingly, intracerebrally inoculation of hamsters with UPrP(Sc) did not cause clinical signs of prion disease even after 270 days, suggesting it differs in its pathogenic properties from brain PrP(Sc). We propose that the detection of UPrP(Sc) can be used to diagnose humans and animals incubating prion diseases, as well as to increase our understanding on the metabolism of PrP(Sc) in vivo.     

Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions

The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.     

Scrapie Agent (Strain 263K) can transmit disease via the oral route after persistence in soil over years

The persistence of infectious biomolecules in soil constitutes a substantial challenge. This holds particularly true with respect to prions, the causative agents of transmissible spongiform encephalopathies (TSEs) such as scrapie, bovine spongiform encephalopathy (BSE), or chronic wasting disease (CWD). Various studies have indicated that prions are able to persist in soil for years without losing their pathogenic activity. Dissemination of prions into the environment can occur from several sources, e.g., infectious placenta or amniotic fluid of sheep. Furthermore, environmental contamination by saliva, excrements or non-sterilized agricultural organic fertilizer is conceivable. Natural transmission of scrapie in the field seems to occur via the alimentary tract in the majority of cases, and scrapie-free sheep flocks can become infected on pastures where outbreaks of scrapie had been observed before. These findings point to a sustained contagion in the environment, and notably the soil. By using outdoor lysimeters, we simulated a contamination of standard soil with hamster-adapted 263K scrapie prions, and analyzed the presence and biological activity of the soil-associated PrP(Sc) and infectivity by Western blotting and hamster bioassay, respectively. Our results showed that 263K scrapie agent can persist in soil at least over 29 months. Strikingly, not only the contaminated soil itself retained high levels of infectivity, as evidenced by oral administration to Syrian hamsters, but also feeding of aqueous soil extracts was able to induce disease in the reporter animals. We could also demonstrate that PrP(Sc) in soil, extracted after 21 months, provides a catalytically active seed in the protein misfolding cyclic amplification (PMCA) reaction. PMCA opens therefore a perspective for considerably improving the detectability of prions in soil samples from the field.     

Propagation of prion strains through specific conformers of the prion protein.

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.     

Novel assay with fluorescence-labelled PrP peptides for differentiating L-type atypical and classical BSEs, and scrapie

Characteristic differences of prions may account for the conformational diversity of the pathogenic isoform of prion protein (PrP(Sc)). Here, we applied a protein detection procedure by using fluorescent-labelled peptides for detecting PrP(Sc). Five prion protein (PrP) related peptides were found to change significantly their fluorescent intensities with prion-affected animal samples. Their reactivity was different among atypical L-BSE, classical BSE and scrapie. The pull-down assay revealed that they precipitated PrP(Sc) specifically. These findings suggest that fluorescent intensity changes depend on peptide-PrP(Sc) binding. This novel approach may distinguish the fine structural differences in PrP(Sc), which were not detected by the pull-down assay.     

Dual nature of the infectious prion protein revealed by high pressure

Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrP(Sc)) and purified prion fibrils were heated or pressurized at 800 megapascals and 60 degrees C for 2 h in different buffers and in water. Prion proteins (PrP) were analyzed for their proteinase K resistance in immunoblots and for their infectivity in hamster bioassays. A notable decrease in the proteinase K resistance of unpurified prion proteins, probably because of pressure-induced changes in the protein conformation of native PrP(Sc) or the N-truncated PrP-(27-30), could be demonstrated when pressurized at initially neutral conditions in several buffers and in water but not in a slightly acidic pH. A subsequent 6-7 log(10) reduction of infectious units/g in phosphate-buffered saline buffer, pH 7.4, was found. The proteinase K-resistant core was also not detectable after purification of prions extracted from pressurized samples, confirming pressure effects at the level of the secondary structure of prion proteins. However, opposite results were found after pressurizing purified prions, arguing for the existence of pressure-sensitive beta-structures (PrP(Sc)(DeltaPsen)) and extremely pressure-resistant beta-structures (PrP(Sc)(DeltaPres)). Remarkably, after the first centrifugation step at 540,000 x g during isolation, prions remained proteinase K-resistant when pressurized in all tested buffers and in water. It is known that purified fibrils retain infectivity, but the isolated protein (full and N-truncated) behaved differently from native PrP(Sc) under pressure, suggesting a kind of semicrystalline polymer structure.     

The oral secretion of infectious scrapie prions occurs in preclinical sheep with a range of PRNP genotypes

Preclinical sheep with the highly scrapie-susceptible VRQ/VRQ PRNP genotype secrete prions from the oral cavity. In order to further understand the significance of orally available prions, buccal swabs were taken from sheep with a range of PRNP genotypes and analyzed by serial protein misfolding cyclic amplification (sPMCA). Prions were detected in buccal swabs from scrapie-exposed sheep of genotypes linked to high (VRQ/VRQ and ARQ/VRQ) and low (ARR/VRQ and AHQ/VRQ) lymphoreticular system involvement in scrapie pathogenesis. For both groups, the level of prion detection was significantly higher than that for scrapie-resistant ARR/ARR sheep which were kept in the same farm environment and acted as sentinel controls for prions derived from the environment which might contaminate the oral cavity. In addition, sheep with no exposure to the scrapie agent did not contain any measurable prions within the oral cavity. Furthermore, prions were detected in sheep over a wide age range representing various stages of preclinical disease. These data demonstrate that orally available scrapie prions may be a common feature in sheep incubating scrapie, regardless of the PRNP genotype and any associated high-level accumulation of PrP(Sc) within lymphoreticular tissues. PrP(Sc) was present in buccal swabs from a large proportion of sheep with PRNP genotypes associated with relatively low disease penetrance, indicating that subclinical scrapie infection is likely to be a common occurrence. The significance of positive sPMCA reactions was confirmed by the transmission of infectivity in buccal swab extracts to Tg338 mice, illustrating the likely importance of orally available prions in the horizontal transmission of scrapie.     

Scrapie infection of prion protein-deficient cell line upon ectopic expression of mutant prion proteins

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.     

Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease

Conversion of cellular prion protein (PrP(C)) into a pathological conformer (PrP(Sc)) is thought to be promoted by PrP(Sc) in a poorly understood process. Here, we report that in wild-type mice, the expression of PrP(C) rendered soluble and dimeric by fusion to immunoglobulin Fcgamma (PrP-Fc(2)) delays PrP(Sc) accumulation, agent replication, and onset of disease following inoculation with infective prions. In infected PrP-expressing brains, PrP-Fc(2) relocates to lipid rafts and associates with PrP(Sc) without acquiring protease resistance, indicating that PrP-Fc(2) resists conversion. Accordingly, mice expressing PrP-Fc(2) but lacking endogenous PrP(C) are resistant to scrapie, do not accumulate PrP-Fc(2)(Sc), and do not transmit disease to others. These results indicate that various PrP isoforms engage in a complex in vivo, whose distortion by PrP-Fc(2) affects prion propagation and scrapie pathogenesis. The unique properties of PrP-Fc(2) suggest that soluble PrP derivatives may represent a new class of prion replication antagonists.     

Computerized morphometric analysis of pathological prion protein deposition in scrapie-infected hamster brain.

Transmissible spongiform encephalopathies (TSEs or prion diseases) are characterized by a constellation of typical though variable pathological changes in the brain. Deposition of disease-associated abnormal prion protein (PrP(Sc)) is the pathological feature of TSEs most consistent and accessible for quantification. However, the evaluation of PrP(Sc) deposits detected by immunohistochemical techniques has been traditionally based on arbitrarily assigned semiquantitative scores. This approach is limited by its subjectivity and bias, yielding considerable variability. In this study, we used MetaMorph 6.1 image analysis software for quantitative analysis of immunostained PrP(Sc) deposits in the CNS of hamsters infected with the 263K strain of scrapie agent. Computerized morphometric analysis (CMA) allowed unambiguous detection of even minimal amounts of immunostained PrP(Sc). CMA values for intensity of staining and area stained correlated well with semiquantitative scores, providing reproducible quantitative data and objective criteria for analyzing PrP(Sc) deposition. CMA provides a simple and reliable method for improved and consistent diagnosis of TSEs that may also be used to quantify other immunostained biomarkers.     

PrPSc incorporation to cells requires endogenous glycosaminoglycan expression

Many lines of evidence suggest an interaction between glycosaminoglycans (GAGs) and the PrP proteins as well as a possible role for GAGs in prion disease pathogenesis. In this work, we sought to determine whether the PrP-GAG interaction affects the incorporation of PrP(Sc) (the scrapie isoform of PrP) to normal cells. This may be the first step in prion disease pathogenesis. To this effect, we incubated proteinase K-digested hamster scrapie brain homogenates with several lines of Chinese hamster ovary (CHO) cells in the presence or absence of heparin. Our results show that over a large range of PrP(Sc) concentrations the binding of PrP(Sc) to wild type CHO cells, which do not express detectable PrP, was equivalent to the binding of PrP(Sc) to CHO cells overexpressing PrP. A significant part of PrP(Sc) binding to both lines could be inhibited by heparin. Additional evidence that PrP(Sc) binding to cells was dependent on the presence of GAGs could be concluded from the fact that the binding of PrP(Sc) to CHO cells missing GAGs on the cell surface was significantly reduced. Interestingly, preincubation of scrapie brain homogenate with heparin before intraperitoneal inoculation into normal hamsters resulted in a significant delay in prion disease manifestation.     

Detection of infectious prions in urine

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals. PrP(Sc) was detected in approximately 80% of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrP(Sc) concentration in urine is around 10-fold lower than in blood. Interestingly, PrP(Sc) present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.     

Intraepithelial and interstitial deposition of pathological prion protein in kidneys of scrapie-affected sheep

Prions have been documented in extra-neuronal and extra-lymphatic tissues of humans and various ruminants affected by Transmissible Spongiform Encephalopathy (TSE). The presence of prion infectivity detected in cervid and ovine blood tempted us to reason that kidney, the organ filtrating blood derived proteins, may accumulate disease associated PrP(Sc). We collected and screened kidneys of experimentally, naturally scrapie-affected and control sheep for renal deposition of PrP(Sc) from distinct, geographically separated flocks. By performing Western blot, PET blot analysis and immunohistochemistry we found intraepithelial (cortex, medulla and papilla) and occasional interstitial (papilla) deposition of PrP(Sc )in kidneys of scrapie-affected sheep. Interestingly, glomerula lacked detectable signals indicative of PrP(Sc). PrP(Sc) was also detected in kidneys of subclinical sheep, but to significantly lower degree. Depending on the stage of the disease the incidence of PrP(Sc) in kidney varied from approximately 27% (subclinical) to 73.6% (clinical) in naturally scrapie-affected sheep. Kidneys from flocks without scrapie outbreak were devoid of PrP(Sc). Here we demonstrate unexpectedly frequent deposition of high levels of PrP(Sc) in ovine kidneys of various flocks. Renal deposition of PrP(Sc) is likely to be a pre-requisite enabling prionuria, a possible co-factor of horizontal prion-transmission in sheep.     

Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.     

Search for a prion-specific nucleic acid

Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID50) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP(Sc) molecules and ID50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID50 unit is approximately 25 nucleotides in length. In the same fractions, there were approximately 3,000 protease-resistant PrP(Sc) molecules per ID50 unit after accounting for clearance of PrP(Sc) following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of approximately 1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.     

Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.     

Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions

Prions are unconventional infectious agents composed exclusively of misfolded prion protein (PrP(Sc)), which transmits the disease by propagating its abnormal conformation to the cellular prion protein (PrP(C)). A key characteristic of prions is their species barrier, by which prions from one species can only infect a limited number of other species. Here, we report the generation of infectious prions by interspecies transmission of PrP(Sc) misfolding by in vitro PMCA amplification. Hamster PrP(C) misfolded by mixing with mouse PrP(Sc) generated unique prions that were infectious to wild-type hamsters, and similar results were obtained in the opposite direction. Successive rounds of PMCA amplification result in adaptation of the in vitro-produced prions, in a process reminiscent of strain stabilization observed upon serial passage in vivo. Our results indicate that PMCA is a valuable tool for the investigation of cross-species transmission and suggest that species barrier and strain generation are determined by the propagation of PrP misfolding.     

Uptake dynamics of scrapie agent in the intestinal villous epithelium of suckling and weanling Syrian hamsters

In mice, the number of intestinal villous columnar epithelium cells that incorporate abnormal prion protein (PrP(Sc) ) decreases significantly after weaning. In this study, the dynamics of PrP(Sc) uptake during the growth of hamsters were investigated by inoculating scrapie 263K agent orally into suckling and weanling Syrian hamsters and estimating the number of PrP(Sc) -positive villous epithelium cells immunohistochemically. The number of PrP(Sc) -positive cells declined significantly as the hamsters aged. The present results suggest that a tendency toward decline of PrP(Sc) -positive cells with increasing age might be a common phenomenon among the superfamily Muridae.     

Prion removal by nanofiltration under different experimental conditions.

Manufacturing processes used in the production of biopharmaceutical or biological products should be evaluated for their ability to remove potential contaminants, including TSE agents. In the present study, we have evaluated scrapie prion protein (PrP Sc) removal in the presence of different starting materials, using virus removal filters of different pore sizes. Following 75 nm filtration, PrP Sc was detected in the filtrate by Western blot (WB) analysis when a "super-sonicated" microsomal fraction derived from hamster adapted scrapie strain 263K (263K MF) was used as the spike material. In contrast, no PrP Sc was detected when an untreated 263K MF was used. By using spike materials prepared in a manner designed to optimize the particle size distribution within the preparation, only 15 nm filtration was shown to remove PrP Sc to below the limits of detection of the WB assays used under all the experimental conditions. However, infectious PrP Sc was recovered following 15 nm filtration under one experimental condition. The results obtained suggest that the nature of the spike preparation is an important factor in evaluating the ability of filters to remove prions, and that procedures designed to minimize the particle size distribution of the prion spike, such as the "super-sonication" or detergent treatments described herein, should be used for the preparation of the spike materials.