Prion Interaction with the 37-kDa/67-kDa Laminin Receptor on Enterocytes as a Cellular Model for Intestinal Uptake of Prions

Enterocytes, a major cell population of the intestinal epithelium, represent one possible barrier to the entry of prions after oral exposure. We established a cell culture system employing enterocytes from different species to study alimentary prion interaction with the 37-kDa/67-kDa laminin receptor LRP/LR. Human, bovine, porcine, ovine, and cervid enterocytes were cocultured with brain homogenates from cervid, sheep, and cattle suffering from chronic wasting disease (CWD), scrapie, and bovine spongiform encephalopathy (BSE), respectively. PrP^CWD, ovine PrP^Sc, and PrP^BSE all colocalized with LRP/LR on human enterocytes. PrP^CWD failed to colocalize with LRP/LR on bovine, porcine, and ovine enterocytes. Ovine PrP^Sc colocalized with the receptor on bovine enterocytes, but failed to colocalize with LRP/LR on cervid and porcine enterocytes. PrP^BSE failed to colocalize with the receptor on cervid and ovine enterocytes. These data suggest possible oral transmissibility of CWD and sheep scrapie to humans and may confirm the oral transmissibility of BSE to humans, resulting in zoonotic variant Creutzfeldt-Jakob disease. CWD might not be transmissible to cattle, pigs, and sheep. Sheep scrapie might have caused BSE, but may not cause transmissible spongiform encephalopathy in cervids and pigs. BSE may not be transmissible to cervids. Our data recommend the enterocyte model system for further investigations of the intestinal pathophysiology of alimentary prion infections.     

Novel Aspects of Prions,Their Receptor Molecules,and Innovative Approaches for TSE Therapy

1. Prion diseases are a group of rare, fatal neurodegenerative diseases, also known as transmissible spongiform encephalopathies (TSEs), that affect both animals and humans and include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt–Jakob disease (CJD) in humans. TSEs are usually rapidly progressive and clinical symptoms comprise dementia and loss of movement coordination due to the accumulation of an abnormal isoform (PrP^Sc) of the host-encoded prion protein (PrP^c). 2. This article reviews the current knowledge on PrP^c and PrP^Sc, prion replication mechanisms, interaction partners of prions, and their cell surface receptors. Several strategies, summarized in this article, have been investigated for an effective antiprion treatment including development of a vaccination therapy and screening for potent chemical compounds. Currently, no effective treatment for prion diseases is available. 3. The identification of the 37 kDa/67 kDa laminin receptor (LRP/LR) and heparan sulfate as cell surface receptors for prions, however, opens new avenues for the development of alternative TSE therapies.     

Experimental transmission of bovine spongiform encephalopathy to European red deer (<Emphasis Type="Italic">Cervus elaphus elaphus</Emphasis>)

Background

Bovine spongiform encephalopathy (BSE), a member of the transmissible spongiform encephalopathies (TSE), primarily affects cattle. Transmission is via concentrate feed rations contaminated with infected meat and bone meal (MBM). In addition to cattle, other food animal species are susceptible to BSE and also pose a potential threat to human health as consumption of infected meat products is the cause of variant Creutzfeldt-Jakob disease in humans, which is invariably fatal. In the UK, farmed and free ranging deer were almost certainly exposed to BSE infected MBM in proprietary feeds prior to legislation banning its inclusion. Therefore, although BSE has never been diagnosed in any deer species, a possible risk to human health remains via ingestion of cervine products. Chronic wasting disease (CWD), also a TSE, naturally infects several cervid species in North America and is spreading rapidly in both captive and free-ranging populations.

Results

Here we show that European red deer (Cervus elaphus elaphus) are susceptible to intra-cerebral (i/c) challenge with BSE positive cattle brain pool material resulting in clinical neurological disease and weight loss by 794–1290 days and the clinical signs are indistinguishable to those reported in deer with CWD. Spongiform changes typical of TSE infections were present in brain and accumulation of the disease-associated abnormal prion protein (PrP^d) was present in the central and peripheral nervous systems, but not in lymphoid or other tissues. Western immunoblot analysis of brain material showed a similar glycosylation pattern to that of BSE derived from infected cattle and experimentally infected sheep with respect to protease-resistant PrP isoforms. However, the di-, mono- and unglycosylated bands migrated significantly (p < 0.001) further in the samples from the clinically affected deer when compared to BSE infected brains of cattle and sheep.

Conclusion

This study shows that deer are susceptible to BSE by intra-cerebral inoculation and display clinical signs and vacuolar pathology that are similar to those of CWD. These findings highlight the importance of preventing the spread to Europe of CWD from North America as this may necessitate even more extensive testing of animal tissues destined for human consumption within the EU. Although the absence of PrP^d in lymphoid and other non-neurological tissues potentially limits the risk of transmission to humans, the replication of TSE agents in peripheral tissues following intra-cerebral challenge is often limited. Thus the assessment of risk posed by cervine BSE as a human pathogen or for environmental contamination should await the outcome of ongoing oral challenge experiments.

     

Prion transmission: Prion excretion and occurrence in the environment

Prion diseases range from being highly infectious, for example scrapie and CWD, which show facile transmission between susceptible individuals, to showing negligible horizontal transmission, such as BSE and CJD, which are spread via food or iatrogenically, respectively. Scrapie and CWD display considerable in vivo dissemination, with PrP^Sc and infectivity being found in a range of peripheral tissues. This in vivo dissemination appears to facilitate the recently reported excretion of prion through multiple routes such as from skin, feces, urine, milk, nasal secretions, saliva and placenta. Furthermore, excreted scrapie and CWD agent is detected within environmental samples such as water and on the surfaces of inanimate objects. The cycle of “uptake of prion from the environment—widespread in vivo prion dissemination—prion excretion—prion persistence in the environment” is likely to explain the facile transmission and maintenance of these diseases within wild and farmed populations over many years.Key words: prion, PrP, excretion, secretion, transmission     

Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies

The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP^Sc amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP^Sc amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP^Sc amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP^C-to-PrP^Sc conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.     

A novel anti‐prion protein monoclonal antibody and its single‐chain fragment variable derivative with ability to inhibit abnormal prion protein accumulation in cultured cells

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121–231. Both mAbs were cross‐reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137–143 of MoPrP and buried in PrP^C expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP^Sc in cultured scrapie‐infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132–217 and this epitope was exposed on the cell surface PrP^C. mAb T2 showed an excellent inhibitory effect on PrP^Sc accumulation in vitro at a 50% inhibitory concentration of 0.02 μg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2‐producing N2a58 cells induced a clear inhibitory effect on PrP^Sc accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP^Sc accumulation.     

Prion transmission

Prion diseases range from being highly infectious, for example scrapie and CWD which show facile transmission between susceptible individuals, to showing negligible horizontal transmission, such as BSE and CJD which are spread via food or iatrogenically respectively. Scrapie and CWD display considerable in vivo dissemination, with PrP^Sc and infectivity being found in a range of peripheral tissues. This in vivo dissemination appears to facilitate the recently reported excretion of prion through multiple routes such as from skin, faeces, urine, milk, nasal secretions, saliva and placenta. Furthermore, excreted scrapie and CWD agent is detected within environmental samples such as water and on the surfaces of inanimate objects. The cycle of ‘uptake of prion from the environment - widespread in vivo prion dissemination - prion excretion - prion persistence in the environment’ is likely to explain the facile transmission and maintenance of these diseases within wild and farmed populations over many years.     

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background

Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrP^Sc, a protease-resistant misfolded isoform of the cellular prion protein PrP^C. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrP^Sc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP^Sc in BSE-affected cattle therefore remains unknown.

Methodology/Principal Findings

We report here that PrP^Sc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP^Sc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP^Sc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP^Sc in blood was not substantiated in the BSE-affected cattle examined.

Conclusions/Significance

The distribution of PrP^Sc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP^Sc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.     

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.     

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrP^C, by its infectious conformation, PrP^Sc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrP^Sc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrP^Sc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.     

Synthetic prions with novel strain-specified properties

Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP^Sc. Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP^Sc were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrP^C to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties.     

Protease-Sensitive Synthetic Prions

Prions arise when the cellular prion protein (PrP^C) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP^Sc. Frequently, PrP^Sc is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP^Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600–750 days in Tg4053 mice, which exhibited sPrP^Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP^Sc.     

Transmission barriers for bovine, ovine, and human prions in transgenic mice

Transgenic (Tg) mice expressing full-length bovine prion protein (BoPrP) serially propagate bovine spongiform encephalopathy (BSE) prions without posing a transmission barrier. These mice also posed no transmission barrier for Suffolk sheep scrapie prions, suggesting that cattle may be highly susceptible to some sheep scrapie strains. Tg(BoPrP) mice were also found to be susceptible to prions from humans with variant Creutzfeldt-Jakob disease (CJD); on second passage in Tg(BoPrP) mice, the incubation times shortened by 30 to 40 days. In contrast, Tg(BoPrP) mice were not susceptible to sporadic, familial, or iatrogenic CJD prions. While the conformational stabilities of bovine-derived and Tg(BoPrP)-passaged BSE prions were similar, the stability of sheep scrapie prions was higher than that found for the BSE prions but lower if the scrapie prions were passaged in Tg(BoPrP) mice. Our findings suggest that BSE prions did not arise from a sheep scrapie strain like the one described here; rather, BSE prions may have arisen spontaneously in a cow or by passage of a scrapie strain that maintains its stability upon passage in cattle. It may be possible to distinguish BSE prions from scrapie strains in sheep by combining conformational stability studies with studies using novel Tg mice expressing a chimeric mouse-BoPrP gene. Single-amino-acid substitutions in chimeric PrP transgenes produced profound changes in incubation times that allowed us to distinguish prions causing BSE from those causing scrapie.     

The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

Prions are unconventional infectious agents thought to be primarily composed of PrP^Sc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP^Sc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP^Sc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP^Sc aggregates from PrP^C. The distribution of PrP^Sc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP^Sc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP^Sc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.     

Direct Detection of Disease Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N Terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.Key Words: BSE, scrapie and vCJD Diagnosis, DELFIA®, novel N terminal antibodies     

Infrared Microspectroscopy Detects Protein Misfolding Cyclic Amplification (PMCA)-induced Conformational Alterations in Hamster Scrapie Progeny Seeds

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP^C) into Proteinase K-resistant, infectious PrP particles (PrP^TSE), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP^C substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP^C substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.     

Evaluation of the Possible Transmission of BSE and Scrapie to Gilthead Sea Bream (Sparus aurata)

In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrP^Sc) of the host-encoded cellular prion protein (PrP^C). While the precise mechanism of the PrP^C to PrP^Sc conversion is not understood, it is clear that host PrP^C expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. Here we show that while gilthead sea bream (Sparus aurata) orally challenged with brain homogenates prepared either from a BSE infected cow or from scrapie infected sheep developed no clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. The control groups, fed with brains from uninfected animals, showed no such signs. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in the ones challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrP^Sc, or of a prion disease developing in farmed fish is alarming and requires further evaluation.     

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

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

Subclinical prion infection

Prion diseases are transmissible neurodegenerative disorders that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt–Jakob disease (CJD) in humans. The principal component of the infectious agent responsible for these diseases appears to be an abnormal isoform of the host-encoded prion protein (PrP), designated PrP^Sc. Prion diseases are transmissible to the same or different mammalian species by inoculation with, or dietary exposure to, infected tissues. Although scrapie in sheep has been recognized for over 200 years, it is the recent epidemic of BSE that has centred much public and scientific attention on these neurodegenerative diseases. The occurrence of variant CJD in humans and the experimental confirmation that it is caused by the same prion strain as BSE has highlighted the need for intensive study into the pathogenesis of these diseases and new diagnostic and therapeutic approaches. The existence and implications of subclinical forms of prion disease are discussed.     

Prion Protein—Antibody Complexes Characterized by Chromatography-Coupled Small-Angle X-Ray Scattering

Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP^C) into a β-sheet-rich disease-causing isoform (PrP^Sc) is the key molecular event in the formation of PrP^Sc aggregates. The molecular mechanisms underlying the PrP^C-to-PrP^Sc conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP^Sc in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23–230)] and N-terminally truncated recPrP(89–230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP^C into PrP^Sc.