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1.  Prion Protein-Specific Antibodies that Detect Multiple TSE Agents with High Sensitivity 
PLoS ONE  2014;9(3):e91143.
This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94–233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays.
doi:10.1371/journal.pone.0091143
PMCID: PMC3946747  PMID: 24608105
2.  EU-Approved Rapid Tests for Bovine Spongiform Encephalopathy Detect Atypical Forms: A Study for Their Sensitivities 
PLoS ONE  2012;7(9):e43133.
Since 2004 it become clear that atypical bovine spongiform encephalopthies (BSEs) exist in cattle. Whenever their detection has relied on active surveillance plans implemented in Europe since 2001 by rapid tests, the overall and inter-laboratory performance of these diagnostic systems in the detection of the atypical strains has not been studied thoroughly to date. To fill this gap, the present study reports on the analytical sensitivity of the EU-approved rapid tests for atypical L- and H-type and classical BSE in parallel. Each test was challenged with two dilution series, one created from a positive pool of the three BSE forms according to the EURL standard method of homogenate preparation (50% w/v) and the other as per the test kit manufacturer's instructions. Multilevel logistic models and simple logistic models with the rapid test as the only covariate were fitted for each BSE form analyzed as directed by the test manufacturer's dilution protocol. The same schemes, but excluding the BSE type, were then applied to compare test performance under the manufacturer's versus the water protocol. The IDEXX HerdChek ® BSE-scrapie short protocol test showed the highest sensitivity for all BSE forms. The IDEXX® HerdChek BSE-scrapie ultra short protocol, the Prionics® - Check WESTERN and the AJ Roboscreen® BetaPrion tests showed similar sensitivities, followed by the Roche® PrionScreen, the Bio-Rad® TeSeE™ SAP and the Prionics® - Check PrioSTRIP in descending order of analytical sensitivity. Despite these differences, the limit of detection of all seven rapid tests against the different classes of material set within a 2 log10 range of the best-performing test, thus meeting the European Food Safety Authority requirement for BSE surveillance purposes. These findings indicate that not many atypical cases would have been missed surveillance since 2001 which is important for further epidemiological interpretations of the sporadic character of atypical forms.
doi:10.1371/journal.pone.0043133
PMCID: PMC3439472  PMID: 22984410
3.  Detection of Prion Protein Particles in Blood Plasma of Scrapie Infected Sheep 
PLoS ONE  2012;7(5):e36620.
Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.
doi:10.1371/journal.pone.0036620
PMCID: PMC3342177  PMID: 22567169
4.  Four Independent Molecular Prion Protein Parameters for Discriminating New Cases of C, L, and H Bovine Spongiform Encephalopathy in Cattle▿ 
Journal of Clinical Microbiology  2011;49(8):3026-3028.
In anticipation of the emergence of more variants of bovine spongiform encephalopathy (BSE), a semiquantitative display of the following four independent molecular diagnostic prion parameters was designed: N terminus, proteinase K (PK) resistance, glycoprofile, and mixed population. One H BSE case, three L BSE cases, six C BSE cases, and one unusual classical BSE (C BSE) case are reported.
doi:10.1128/JCM.01102-11
PMCID: PMC3147744  PMID: 21677067
5.  Distinct Proteinase K-Resistant Prion Protein Fragment in Goats with No Signs of Disease in a Classical Scrapie Outbreak▿† 
Journal of Clinical Microbiology  2011;49(6):2109-2115.
Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrPres) in a highly scrapie-affected goat flock in Greece. The PrPres profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrPres fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrPres phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.
doi:10.1128/JCM.02033-10
PMCID: PMC3122744  PMID: 21450953
6.  Variably Protease-Sensitive Prionopathy: A New Sporadic Disease of the Prion Protein 
Annals of neurology  2010;68(2):162-172.
Objective
The objective of the study is to report 2 new genotypic forms of protease-sensitive prionopathy (PSPr), a novel prion disease described in 2008, in 11 subjects all homozygous for valine at codon 129 of the prion protein (PrP) gene (129VV). The 2 new PSPr forms affect individuals who are either homozygous for methionine (129MM) or heterozygous for methionine/valine (129MV).
Methods
Fifteen affected subjects with 129MM, 129MV, and 129VV underwent comparative evaluation at the National Prion Disease Pathology Surveillance Center for clinical, histopathologic, immunohistochemical, genotypical, and PrP characteristics.
Results
Disease duration (between 22 and 45 months) was significantly different in the 129VV and 129MV subjects. Most other phenotypic features along with the PrP electrophoretic profile were similar but distinguishable in the 3 129 genotypes. A major difference laid in the sensitivity to protease digestion of the disease-associated PrP, which was high in 129VV but much lower, or altogether lacking, in 129MV and 129MM. This difference prompted the substitution of the original designation with “variably protease-sensitive prionopathy” (VPSPr). None of the subjects had mutations in the PrP gene coding region.
Interpretation
Because all 3 129 genotypes are involved, and are associated with distinguishable phenotypes, VPSPr becomes the second sporadic prion protein disease with this feature after Creutzfeldt-Jakob disease, originally reported in 1920. However, the characteristics of the abnormal prion protein suggest that VPSPr is different from typical prion diseases, and perhaps more akin to subtypes of Gerstmann-Sträussler-Scheinker disease.
doi:10.1002/ana.22094
PMCID: PMC3032610  PMID: 20695009
7.  Co-existence of scrapie prion protein types 1 and 2 in sporadic Creutzfeldt–Jakob disease: its effect on the phenotype and prion-type characteristics 
Brain  2009;132(10):2643-2658.
Five phenotypically distinct subtypes have been identified in sporadic Creutzfeldt–Jakob disease (sCJD), based on the methionine/valine polymorphic genotype of codon 129 of the prion protein (PrP) gene and the presence of either one of the two protease K-resistant scrapie prion protein (PrPSc) types identified as 1 and 2. The infrequent co-existence of both PrPSc types in the same case has been known for a long time. Recently, it has been reported, using type-specific antibodies, that the PrPSc type 1 is present in all cases of sCJD carrying PrPSc type 2. The consistent co-occurrence of both PrPSc types complicates the diagnosis and the current classification of sCJD, and has implications for the pathogenesis of naturally occurring prion diseases. In the present study, we investigated the prevalence of PrPSc types 1 and 2 co-occurrence, along with its effects on the disease phenotype and PrPSc strain characteristics, comparatively analysing 34 cases of sCJD, all methionine homozygous at codon 129 of the PrP gene (sCJDMM). To minimize overestimating the prevalence of the sCJDMM cases carrying PrPSc types 1 and 2 (sCJDMM1-2), we used proteinase K concentrations designed to hydrolyse all fragments resulting from an incomplete digestion, while preserving the protease-resistant PrPSc core. Furthermore, we used several antibodies to maximize the detection of both PrPSc types. Our data show that sCJDMM cases associated exclusively with either PrPSc type 1 (sCJDMM1) or PrPSc type 2 (sCJDMM2) do exist; we estimate that they account for approximately 56% and 5% of all the sCJDMM cases, respectively; while in 39% of the cases, both PrPSc types 1 and 2 are present together (sCJDMM1-2) either mixed in the same anatomical region or separate in different regions. Clinically, sCJDMM1-2 had an average disease duration intermediate between the other two sCJDMM subtypes. The histopathology was also intermediate, except for the cerebellum where it resembled that of sCJDMM1. These features, along with the PrP immunostaining pattern, offer a diagnostic clue. We also observed a correlation between the disease duration and the prevalence of PrPSc type 2 and sCJDMM2 phenotypes. The use of different antibodies and of the conformational stability immunoassay indicated that the co-existence of types 1 and 2 in the same anatomical region may confer special conformational characteristics to PrPSc types 1 and 2. All of these findings indicate that sCJDMM1-2 should be considered as a separate entity at this time.
doi:10.1093/brain/awp196
PMCID: PMC2766234  PMID: 19734292
prion protein; prion disease; co-existence; conformation; sporadic Creutzfeldt–Jakob disease
8.  Incidence and spectrum of sporadic Creutzfeldt–Jakob disease variants with mixed phenotype and co-occurrence of PrPSc types: an updated classification 
Acta Neuropathologica  2009;118(5):659-671.
Six subtypes of sporadic Creutzfeldt–Jakob disease with distinctive clinico-pathological features have been identified largely based on two types of the abnormal prion protein, PrPSc, and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein. The existence of affected subjects showing mixed phenotypic features and concurrent PrPSc types has been reported but with inconsistencies among studies in both results and their interpretation. The issue currently complicates diagnosis and classification of cases and also has implications for disease pathogenesis. To explore the issue in depth, we carried out a systematic regional study in a large series of 225 cases. PrPSc types 1 and 2 concurrence was detected in 35% of cases and was higher in MM than in MV or VV subjects. The deposition of either type 1 or 2, when concurrent, was not random and always characterized by the coexistence of phenotypic features previously described in the pure subtypes. PrPSc type 1 accumulation and related pathology predominated in MM and MV cases, while the type 2 phenotype prevailed in VVs. Neuropathological examination best identified the mixed types 1 and 2 features in MMs and most MVs, and also uniquely revealed the co-occurrence of pathological variants sharing PrPSc type 2. In contrast, molecular typing best detected the concurrent PrPSc types in VV subjects and MV cases with kuru plaques. The present data provide an updated disease classification and are of importance for future epidemiologic and transmission studies aimed to identify etiology and extent of strain variation in sporadic Creutzfeldt–Jakob disease.
doi:10.1007/s00401-009-0585-1
PMCID: PMC2773124  PMID: 19718500
Prion protein; Brain mapping; Molecular typing; Neurodegeneration; Classification
9.  Molecular Discrimination of Atypical Bovine Spongiform Encephalopathy Strains from a Geographical Region Spanning a Wide Area in Europe† ▿  
Journal of Clinical Microbiology  2007;45(6):1821-1829.
Transmissible spongiform encephalopathy strains can be differentiated by their behavior in bioassays and by molecular analyses of the disease-associated prion protein (PrP) in a posttranslationally transformed conformation (PrPSc). Until recently, isolates from cases of bovine spongiform encephalopathy (BSE) appeared to be very homogeneous. However, a limited number of atypical BSE isolates have recently been identified upon analyses of the disease-associated proteinase K (PK) resistance-associated moiety of PrPSc (PrPres), suggesting the existence of at least two additional BSE PrPres variants. These are defined here as the H type and the L type, according to the higher and lower positions of the nonglycosylated PrPres band in Western blots, respectively, compared to the position of the band in classical BSE (C-type) isolates. These molecular PrPres variants, which originated from six different European countries, were investigated together. In addition to the migration properties and glycosylation profiles (glycoprofiles), the H- and L-type isolates exhibited enhanced PK sensitivities at pH 8 compared to those of the C-type isolates. Moreover, H-type BSE isolates exhibited differences in the binding of antibodies specific for N- and more C-terminal PrP regions and principally contained two aglycosylated PrPres moieties which can both be glycosylated and which is thus indicative of the existence of two PrPres populations or intermediate cleavage sites. These properties appear to be consistent within each BSE type and independent of the geographical origin, suggesting the existence of different BSE strains in cattle. The choice of three antibodies and the application of two pHs during the digestion of brain homogenates provide practical and diverse tools for the discriminative detection of these three molecular BSE types and might assist with the recognition of other variants.
doi:10.1128/JCM.00160-07
PMCID: PMC1933055  PMID: 17442800
10.  Transmission of New Bovine Prion to Mice 
Emerging Infectious Diseases  2006;12(7):1125-1128.
We previously reported that cattle were affected by a prion disorder that differed from bovine spongiform encephalopathy (BSE) by showing distinct molecular features of disease-associated protease-resistant prion protein (PrPres). We show that intracerebral injection of such isolates into C57BL/6 mice produces a disease with preservation of PrPres molecular features distinct from BSE.
doi:10.3201/eid1207.060107
PMCID: PMC3291063  PMID: 16836832
Bovine spongiform encephalopathy; prion; cattle; dispatch
11.  Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease‡  
Journal of Virology  2006;80(2):596-604.
Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrPCWD) was used as an indicator of CWD infection. Although no PrPCWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrPCWD-positive clone out of 51. This clone, designated MDBCWD, has maintained stable PrPCWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrPCWD-positive subclones out of 30, one of which was designated MDBCWD2. The MDBCWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrPCWD accumulation in MDBCWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrPCWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo.
doi:10.1128/JVI.80.2.596-604.2006
PMCID: PMC1346862  PMID: 16378962
12.  Extending the CD4+ T-Cell Epitope Specificity of the Th1 Immune Response to an Antigen Using a Salmonella enterica Serovar Typhimurium Delivery Vehicle 
Infection and Immunity  2000;68(6):3079-3089.
We analyzed the CD4 T-cell immunodominance of the response to a model antigen (Ag), MalE, when delivered by an attenuated strain of Salmonella enterica serovar Typhimurium (SL3261*pMalE). Compared to purified MalE Ag administered with adjuvant, the mapping of the peptide-specific proliferative responses showed qualitative differences when we used the Salmonella vehicle. We observed the disappearance of one out of eight MalE peptides' T-cell reactivity upon SL3261*pMalE immunization, but this phenomenon was probably due to a low level of T-cell priming, since it could be overcome by further immunization. The most striking effect of SL3261*pMalE administration was the activation and stimulation of new MalE peptide-specific T-cell responses that were silent after administration of purified Ag with adjuvant. Ag presentation assays performed with MalE-specific T-cell hybridomas showed that infection of Ag-presenting cells by this intracellular attenuated bacterium did not affect the processing and presentation of the different MalE peptides by major histocompatibility complex (MHC) class II molecules and therefore did not account for immunodominance modulation. Thus, immunodominance of the T-cell response to microorganisms is governed not only by the frequency of the available T-cell repertoire or the processing steps in Ag-presenting cells that lead to MHC presentation but also by other parameters probably related to the infectious process and to the bacterial products. Our results indicate that, upon infection by a microorganism, the specificity of the T-cell response induced against its Ags can be much more effective than with purified Ags and that it cannot completely be mimicked by purified Ags administered with adjuvant.
PMCID: PMC97535  PMID: 10816447

Results 1-12 (12)