Earliest detection of prion seeding activity in blood of human PRP transgenic mice infected with vCJD

International audienc

Assessment of prion infectivity distribution in primate blood among cell fractions

International audienceBackground: Probable interhuman transmission of vCJD through blood and derived products has been reported in several cases in Great Britain, highlighting the necessity to secure transfusion against prion risk. Experimental models suggest an equal distribution of infectivity among plasma and white cells, but the cellular populations supporting infectivity remain to be clearly identified. Objectives: Experimental BSE/vCJD infection of cynomolgus macaque constitutes a model of choice to better assess the distribution of prion infectivity among the different blood cell populations. Methods: Monkeys were experimentally infected with BSE by the oral route, or with primate-adapted BSE by the intravenous route (secondary and tertiary passages). In the context of the PrionBloodPrimate project funded by Alliance BioSecure Foundation, blood was sampled and fractionated according to an adapted protocol to concentrate white cells. Different cell populations were then sorted with the Foundation’s secured cell sorter (Influx). Infectivity of whole blood, plasma and the different cell fractions will be assessed by inoculation of transgenic mice overexpressing human PrPMet129 (tg650). Results: Primates were selected for their presence of peripheral infectivity according to the presence of PrPres in biopsied inguinal lymph nodes. Corresponding granulocytes, monocytes, lymphocytes and dendritic cells fractions were separated and enriched with purity around 90%. tg650 mice that demonstrate a high susceptibility to vCJD and allow endpoint titration of infectivity within relatively short delays, have been inoculated with these samples and the first results will be available for the congress Prion 2009. Discussion: With detailed information about donor primates (incubation period, presence of peripheral replication or clinical signs), the efficient actors are now gathered to evaluate the distribution of infectivity among the different components of these unique samples, to better assess the risk of transmission of prion by blood transfusion

Earliest detection of prion seeding activity in blood of human PRP transgenic mice infected with vCJD

International audienc

Détection précoce des prions responsables du variant de la maladie de Creutzfeldt- Jakob dans le sang de souris humanisées

International audienc

Early stage prion assembly involves two subpopulations with different quaternary structures and a secondary templating pathway

International audienceThe dynamics of aggregation and structural diversification of misfolded, host-encoded proteins in neurodegenerative diseases are poorly understood. In many of these disorders, including Alzheimer’s, Parkinson’s and prion diseases, the misfolded proteins are self-organized into conformationally distinct assemblies or strains. The existence of intrastrain structural heterogeneity is increasingly recognized. However, the underlying processes of emergence and coevolution of structurally distinct assemblies are not mechanistically understood. Here, we show that early prion replication generates two subsets of structurally different assemblies by two sequential processes of formation, regardless of the strain considered. The first process corresponds to a quaternary structural convergence, by reducing the parental strain polydispersity to generate small oligomers. The second process transforms these oligomers into larger ones, by a secondary autocatalytic templating pathway requiring the prion protein. This pathway provides mechanistic insights into prion structural diversification, a key determinant for prion adaptation and toxicity

Correlation between Bioassay and Protein Misfolding Cyclic Amplification for Variant Creutzfeldt-Jakob Disease Decontamination Studies

International audienceTo date, approximately 500 iatrogenic Creutzfeldt-Jakob disease cases have been reported worldwide, most of them resulting from cadaveric dura mater graft and from the administration of prion-contaminated human growth hormone. The unusual resistance of prions to decontamination processes, their large tissue distribution, and the uncertainty about the prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the general population lead to specific recommendations regarding identification of tissue at risk and reprocessing of reusable medical devices, including the use of dedicated treatment for prion inactivation. We previously described an in vitro assay, called Surf-PMCA, which allowed us to classify prion decontamination treatments according to their efficacy on vCJD prions by monitoring residual seeding activity (RSA). Here, we used a transgenic mouse line permissive to vCJD prions to study the correlation between the RSA measured in vitro and the in vivo infectivity. Implantation in mouse brains of prion-contaminated steel wires subjected to different decontamination procedures allows us to demonstrate a good concordance between RSA measured by Surf-PMCA (in vitro) and residual infectivity (in vivo). These experiments emphasize the strength of the Surf-PMCA method as a rapid and sensitive assay for the evaluation of prion decontamination procedures and also confirm the lack of efficacy of several marketed reagents on vCJD prion decontamination

Preclinical Detection of Variant CJD and BSE Prions in Blood

International audienceThe emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q(171) PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients

PrP^res in PMCA reactions seeded with WBC or BC from a vCJD affected patients and healthy controls.

<p>(<b>A</b>) WBCs from a French vCJD affected patient (vCJD-WBC) and healthy controls (H-WBC) were used to seed serial PMCA amplification (six rounds). PMCA controls included unseeded reactions (no seed). (<b>B</b>) Similarly, nine human buffy coat samples (received from the MRC Prion Unit (London, UK) were used to seed serial PMCA amplification (six rounds). The panel included three vCJD affected patients (sample 1, 3 and 8) and six healthy controls. A vCJD brain homogenate (10%, 10⁻⁸ diluted) was used as positive amplification control. In all cases brain homogenate from ovine PrP transgenic mice (ARQ variant) was used as substrate. PMCA products were analyzed by Western blot (WB) for the presence of abnormal PK resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control).</p

vCJD agent detection in the buffy coat of experimentally infected primates.

<p>Eight cynomologus macaques were intravenously challenged with vCJD brain homogenate or blood from a vCJD affected macaque (macaque 6). At different time points of the incubation period, blood was collected and buffy coat prepared. Clinical onset (clin) and time to euthanasia of the animals are indicated (upper label on arrows) as months post inoculation (mpi). The buffy coat samples were used (as homogenates 1/100 diluted in PMCA buffer) to seed PMCA reactions in which brain homogenate from ovine PrP transgenic mouse (ARQ variant) was used as substrate. Each sample was submitted to 6 rounds of amplification each comprising 96 cycles (30 s sonication-30 minutes incubation at 39.5°C) in a Misonix 4000 sonicator. PMCA products were analyzed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE). Samples were received encoded and tested blind. The time point corresponding to blood samples (months post inoculation) that were tested and the results of PrP^res WB detection in PMCA reactions are indicated (under arrow). No positive WB result was observed before the third PMCA round. No additional positive result was observed after 5 PMCA rounds.</p

PrP^res detection in PMCA reactions seeded with white blood cells (WBC) from BSE infected and healthy sheep.

<p>WBC from BSE orally challenged and TSE free control ARQ/ARQ sheep were homogenised and used to seed PMCA reactions. Brain homogenate from ovine PrP transgenic mouse (ARQ variant) was used as PMCA substrate. Each sample was submitted to up to 6 rounds of amplification. Resulting PMCA products were analyzed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control). (<b>A</b>) In BSE orally challenged sheep (ARQ/ARQ), WBC prepared from blood collected at different time points (indicated as months post inoculation: mpi) of the incubation period were tested. The first clinical signs developed at 20 mpi. (<b>B</b>) BSE affected sheep (3 different individuals-20 mpi) and TSE free controls sheep (breed, genotype and age matched) were submitted to up to 6 PMCA rounds to check the specificity of the amplification.</p