Preclinical Detection of Variant CJD and BSE Prions in Blood

Abstract

The 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 Q171 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.

Factors intrinsic and extrinsic to blood hamper the development of a routine blood test for human prion diseases

“…It also allowed detection of PrP TSE in whole blood collected from CD1 mice that were experimentally infected with the Rocky Mountain Laboratory (RML) prion strain of scrapie, and in whole blood from uninfected CD1 mice that was exogenously spiked with RMLinfected brain homogenate (Tattum et al, 2010a). Furthermore, very recently, PMCA was also used to detect PrP TSE in the white blood cells and buffy coat from BSE-infected sheep and vCJD-infected primates (cynomolgus monkey) during the preclinical and clinical stages of the disease, and from vCJD-affected patients (Lacroux et al, 2014).…”

“…Some of the assays reviewed here, particularly the solidstate matrix assays (Lourenco et al, 2006;Edgeworth et al, 2011;Lacroux et al, 2014), demonstrated that it is possible to detect PrP TSE in the blood of symptomatic vCJD patients. This is a marked advance in the diagnosis of prion diseases, indicating that a blood test is possible, although not yet suitable, for routine use.…”

“…However, based on the fact that PrP TSE from vCJD-infected brain homogenate spiked into whole blood can be detected with good sensitivity at dilution of 10 210 , hypothetically, it should be possible to detect prions in the blood of vCJD patients at the preclinical stage of the disease (Edgeworth et al, 2011). Moreover, given that other blood tests based on different principles showed similar behaviour, but that not all blood samples obtained from clinically affected subjects tested positive for PrP TSE (Terry et al, 2009;Bannach et al, 2012;Jackson et al, 2014;Lacroux et al, 2014), the sensitivity of the tests is unlikely to be the only factor responsible for the failed detection of PrP TSE . Rather, it may be related to the variable presence of factors intrinsic and extrinsic to blood that differentially affect the concentration and/or detection of PrP TSE in blood as compared to other tissues.…”

“…It is also supported by the fact that PrP TSE present in blood is both associated with cellular components and in a cellfree state (Table 1), implying that, due dynamic equilibrium, the ratio of free to associated PrP TSE is continuously subject to change during the disease period. Consequently, the detection systems could detect concentrations of PrP TSE in blood that differ considerably among infected individuals, or in some individuals PrP TSE could even seem absent (Terry et al, 2009;Bannach et al, 2012;Jackson et al, 2014;Lacroux et al, 2014).…”

Bioassay of prion-infected blood plasma in PrP transgenic Drosophila

“…However, the occurrence of vCJD cases caused by administration of prion-infected human blood has highlighted the realistic opportunity for the development of a blood-based diagnostic test for this condition. Recent developments have shown promise with the detection of vCJD-positive blood samples from clinical vCJD cases by immuno-biochemical selection of disease-associated PrP, without the use of PK [42,43], and by detection of PK-resistant PrPSc following PMCA [44]. Since transmissibility is a defining hallmark of prion diseases, it will be important to develop a reasonably rapid and versatile confirmatory prion infectivity bioassay to supplement these biochemical-based prion diagnostic assays.…”